Neutrophil/TGF-β Axis Limits Pathogenicity of Allergen-Specific CD4+ T Cells Notes
Neutrophil/TGF-β Axis Limits Pathogenicity of Allergen-Specific CD4+ T Cells
The intensity and longevity of inflammatory responses to inhaled allergens depend on the balance between effector and regulatory immune responses.
The type of adjuvant used during allergic sensitization significantly affects the nature and longevity of pulmonary inflammation triggered by subsequent allergen reexposure.
TLR ligand adjuvants and house dust extracts prime mixed neutrophilic and eosinophilic inflammation, which is suppressed by multiple daily allergen challenges.
During TLR ligand-mediated allergic sensitization, mice exhibit transient airway neutrophilia, triggering TGF-β release into the airway.
Neutrophil-dependent TGF-β production during sensitization has a delayed, suppressive effect on eosinophilic responses to subsequent allergen challenge.
Neutrophil depletion during sensitization increases proportions of Gata3+CD4+ T cells, leading to stronger eosinophilic inflammation upon transfer to recipient mice.
The neutrophil/TGF-β axis acts during TLR-mediated allergic sensitization to fine-tune allergen-specific CD4+ T cell phenotype and limit their pathogenicity, suggesting a novel immunotherapeutic approach to control eosinophilia in asthma.
Introduction
Allergic asthma is prevalent, chronic inflammatory airway disease characterized by reversible airway obstruction, airway hyperresponsiveness (AHR), and inflammation.
Approximately 8% of the adult US population has asthma; globally, over 300 million people are affected, creating a significant economic and quality of life burden.
The rising prevalence of asthma suggests environmental factors are driving the increase; various environmental exposures are linked to increased asthma incidence.
Asthma is heterogeneous, with different pathologies distinguished by the nature of airway inflammation.
Some patients exhibit eosinophilic inflammation (associated with type 2 immune responses), while others have noneosinophilic asthma with predominant neutrophilia.
Allergen-specific T helper 2 (Th2) and Th17 cells likely drive eosinophilic and neutrophilic inflammation, respectively.
Asthma "endotypes" propose that different forms arise from perturbations of distinct cellular and molecular pathways.
Understanding these pathways may lead to novel therapeutic targets for specific asthma endotypes.
Asthma development involves two phases:
Phase 1 (asymptomatic): Inhaled allergens are taken up by lung dendritic cells, which migrate to regional lymph nodes (LNs) and present allergen-derived peptides to naive T cells, leading to effector T cell differentiation and proliferation.
Phase 2: Reexposure triggers migration of T cells to the lung, where they release cytokines and other molecules, driving airway inflammation.
The microenvironment affects T cell responses; in vitro, IL-12 and IFN-γ induce Th1 cells, IL-4 drives Th2 cells, TGF-β elicits Tregs, and TGF-β with IL-1 or IL-6 induces Th17 differentiation.
Little is known about how local cytokine concentrations are generated in vivo and how adjuvants affect their levels.
Protease adjuvants promote type 2 cytokine-driven, eosinophilic inflammation, while TLR ligands like LPS and flagellin promote mixed neutrophilic and eosinophilic inflammation (Th2 and Th17 responses).
LPS contributes to the allergenicity of house dust mite allergen; household levels of bacteria and endotoxin correlate with asthma prevalence.
Paradoxically, endotoxin exposure, especially early in life in agricultural settings, is associated with protection from developing asthma later in life.
Inhalation of LPS can induce Treg proliferation and suppress asthma-like responses in mice.
Understanding the mechanisms by which LPS promotes allergic sensitization and protects against asthma may lead to novel therapeutic strategies.
Allergic airway inflammation was suppressed by prolonged allergen challenges in mice sensitized to OVA using LPS, but not in mice sensitized using a protease adjuvant.
This suppression depended on acute airway neutrophilia and consequent TGF-β release during the initial LPS-mediated sensitization phase.
Neutrophil depletion during sensitization blocked TGF-β production and prevented suppression of allergic responses to prolonged allergen challenge.
Recombinant mouse TGF-β delivered to the airways during sensitization restored immunosuppression that had been prevented by neutrophil depletion.
Neutrophils in the airway during allergic sensitization did not affect initial Th2 cell development or proliferation but had a lasting impact on suppressing the expansion and activity of Gata3+ Th2 cells.
The neutrophil influx and TGF-β production during allergic sensitization blunted Th2 cell pathogenicity and limited asthma longevity.
Results
Mice sensitized to an allergen using protease adjuvants develop eosinophilic inflammation, while mice sensitized using TLR ligands (LPS or flagellin) develop mixed eosinophilic and neutrophilic inflammation after a single challenge.
Proteases prime predominantly Th2 responses, while LPS primes both Th2 and Th17 responses.
Intracellular staining of CD4+ T cells for Th lineage-specific transcription factors was performed following allergic sensitization and a single OVA challenge.
Mice sensitized to OVA using a mix of proteases from Aspergillus oryzae (ASP/OVA) had high percentages of Th2 cells (Gata3+) and low percentages of Th17 cells (RORγt+).
Mice sensitized using LPS from E. coli (LPS/OVA) had low percentages of Th2 cells and a high proportion of Th17 cells.
No adjuvant-specific differences were seen in the percentages of Foxp3+ Tregs.
Mice were sensitized to OVA using ASP/OVA or LPS/OVA and subjected to various numbers of daily challenges with aerosolized OVA.
Eosinophilia was higher in ASP/OVA-sensitized mice than in LPS/OVA-sensitized animals; in both groups, eosinophil numbers increased with the number of allergen challenges, up to 3 challenges.
Additional challenges increased eosinophils in ASP/OVA-sensitized mice, but these cells declined markedly in LPS/OVA-sensitized animals after 3 challenges.
Neutrophilic inflammation also declined in LPS/OVA-sensitized mice challenged on more than 3 occasions, whereas the relatively low number of neutrophils in ASP/OVA-sensitized mice remained unchanged.
Mice sensitized with ASP/OVA developed robust AHR after a single challenge, and this was sustained even after multiple daily allergen challenges.
LPS/OVA-sensitized mice developed AHR after a single challenge, but it was greatly diminished after multiple allergen challenges.
The declining inflammatory and physiologic responses to prolonged allergen challenge in LPS/OVA-sensitized mice might have been due to the weaker Th2 response or to active suppression by prolonged exposure to the allergen.
Mice sensitized to LPS/OVA and challenged on 2 occasions, 2 weeks apart, responded as vigorously to the second OVA challenge as they did to the first challenge, including their development of AHR.
Continued daily challenges are required for the suppression of allergic responses.
Mice were sensitized with LPS/OVA and challenged on 7 consecutive days, then allowed to rest for 1 week before an additional, final challenge.
Multiple challenges reduced the eosinophilic inflammation, and the 1-week rest period was sufficient for that inflammation to resolve.
Control mice challenged on 7 consecutive days and rested for 1 week, but not rechallenged, had neither robust inflammation nor AHR.
These animals also had very low airway levels of IL-4, IL-5, and IL-17.
Animals that underwent a similar challenge and rest period, but were subsequently rechallenged, displayed multiple asthma-like features, including inflammation, AHR, and cytokine production.
Eosinophils were slightly lower in the group undergoing multiple challenges before the final rechallenge, but this difference was not statistically significant.
Animals retain allergen-responsive effector cells whose actions are temporarily suppressed by continued daily allergen exposures.
Animals sensitized using this LPS/ASP/OVA mixture displayed a similar pattern of eosinophilic inflammation to that seen in mice sensitized to LPS/OVA, with numbers of these cells progressively increasing after 1 or 3 daily challenges but decreasing thereafter.
Although LPS acts as an adjuvant to promote allergen-specific Th2 and Th17 immune responses, it also triggers an immunoregulatory response that becomes dominant in the face of repeated exposure to allergen.
Common house dust contains a mixture of environmental adjuvants, including proteases and TLR ligands, such as LPS.
Animals sensitized with HDE/OVA displayed airway inflammation for up to 3 daily OVA challenges, but these responses declined with additional daily challenges.
Common house dust also contains agents that limit the longevity of allergic responses to inhaled allergens.
Mice were challenged with aerosolized OVA in the same exposure chamber and at the same time; differences must have been due to distinct cellular and molecular pathways triggered by adjuvants at the time of sensitization.
Instillation of LPS/OVA into the airway caused rapid airway inflammation, due almost entirely to neutrophilia, which did not occur in ASP/OVA-treated mice.
Neutrophils recruited to the airway during LPS-mediated allergic sensitization have a regulatory effect on immune responses that only becomes apparent after multiple daily allergen challenges.
Injection of the neutrophil-depleting antibody, anti-Ly6G (clone 1A8), significantly reduced the number of neutrophils in the airspace following LPS inhalation but did not affect macrophage number.
Neutrophil depletion during LPS/OVA sensitization did not significantly affect subsequent responses of mice to a single OVA challenge, although there was a trend toward increased eosinophilia.
Antibody-mediated neutrophil depletion during sensitization did not affect numbers of neutrophils in the airway after allergen challenge, as neutrophil numbers recover within 2 or 3 days of 1A8-mediated depletion.
Depleting neutrophils during sensitization had a dramatic effect on subsequent responses to 6 consecutive daily allergen challenges, with mice displaying a striking and selective increase in eosinophilia compared with mice that received either no antibody or the isotype control (IC) antibody.
Similar findings were observed when we used an anti–Gr-1 antibody, which depletes both neutrophils and monocytes.
Neutrophil depletion during LPS/OVA sensitization also affected physiologic responses to subsequent OVA challenges, as indicated by increased AHR in these mice.
The ability of neutrophil depletion during allergic sensitization to increase asthma-like features during the challenge phase was not unique to the LPS/OVA model of asthma, because we also observed this in models, the HDE/OVA model and another model in which HDE served as both the adjuvant and source of allergens.
Instillation of bacterial flagellin into the airway together with OVA triggered transient neutrophilia, and depletion of neutrophils during flagellin-mediated allergic sensitization heightened responses to subsequent allergen challenge.
Airway neutrophilia during allergic sensitization has a delayed, suppressive effect on eosinophilia and AHR in multiple models of asthma.
Previous studies have shown that mice sensitized to OVA using very small doses of LPS (1 ng) display more sustained responses to OVA challenge than mice sensitized using higher doses (100 ng).
Allergic sensitization using relatively low doses of LPS led to less acute airway neutrophilia than when higher doses of LPS were used.
Including a neutrophil-attracting chemokine together with very low doses of LPS during allergic sensitization might phenocopy the suppressive responses to prolonged allergen challenge seen when higher doses of LPS are used as the adjuvant.
Supplementation of very-low-dose LPS (1 ng) with 0.35 μg of the neutrophil-attracting chemokine, CXCL1, recruited as many neutrophils to the airway as did administration of a 100-fold higher dose of LPS (100 ng) and that there were minimal effects on macrophage numbers.
Inclusion of CXCL1 with low-dose LPS during allergic sensitization did not significantly reduce eosinophilic responses to a single OVA challenge, although there was a trend in that direction.
Including CXCL1 during allergic sensitization did reduce the eosinophilia seen after 6 daily OVA challenges.
Inclusion of CXCL1 with small amounts of LPS during sensitization was not sufficient to drive Th17 responses, as these mice did not have significant neutrophilia after challenge.
Airway neutrophilia during allergic sensitization is both necessary and sufficient to negatively regulate eosinophilic inflammation during prolonged allergen challenge.
Antigen-presenting cells (APCs) are required during the challenge phase of mouse models of asthma; APC actions can be suppressed by prior exposure to a strong initial stimulus.
Neutrophil recruitment to the airway promotes allergen-dependent immunosuppression by affecting APC function.
Neutrophils were depleted prior to LPS inhalation, omitting OVA, which would have stimulated OVA-specific T cells.
Adoptive transfer of in vitro-generated, OVA-specific Th2 cells to the LPS-treated mice, followed by 5 consecutive daily OVA challenges, showed no difference in eosinophilic inflammation between recipients that had undergone prior 1A8-mediated neutrophil depletion and those that had not.
Effects of neutrophils on nonlymphocyte, lung resident cells, such as epithelial cells or dendritic cells, are not responsible for the immunosuppression seen following prolonged allergen challenges.
Mice were sensitized with HDE/OVA, with or without neutrophil depletion, then challenged daily with OVA for 5 days, and intracellular was staining performed on T cells for lineage-specific transcription factors.
Neutrophil depletion did not affect percentages of total CD4+ T cells in the lung, but mice in which neutrophils had been depleted during allergic sensitization had increased percentages of Gata3+ Th2 cells compared with mice that received no antibody or IC antibody.
The increase in Gata3+ Th2 cells is consistent with the increased eosinophilia we had observed in mice that underwent neutrophil depletion during allergic sensitization.
Foxp3– IL-10+ type 1 Tregs made up less than 2% of CD4+ T cells, and their numbers were not significantly affected by neutrophil depletion.
Foxp3+IL-10– Tregs were much more abundant, making up more than 25% of CD4+ T cells, but neutrophil depletion did not affect their numbers.
Tregs from mice that had undergone neutrophil depletion were less effective at suppressing proliferation, suggesting that the increased numbers of eosinophils in neutrophil-depleted mice might stem at least in part from reduced Treg activity.
Neutrophil depletion during sensitization enhanced the pathogenicity of conventional CD4+ T cells.
Naive OVA-specific OT-II T cells were adoptively transferred into C57BL/6 mice before the animals underwent HDE/OVA sensitization, with or without neutrophil depletion.
After 5 daily OVA challenges, total CD4+ T cells were isolated from lungs of each group of mice and transferred into separate groups of recipient mice that had already been sensitized twice with HDE/OVA.
Mice given CD4+ T cells from donors that received either no antibody or IC antibody during sensitization displayed only modest eosinophilia after 3 challenges.
Mice receiving CD4+ T cells from donors undergoing neutrophil depletion during sensitization displayed very robust eosinophilia.
The presence of neutrophils during allergic sensitization curtails the pathogenicity of developing allergen-specific CD4+ T cells, and depleting those neutrophils during sensitization unleashes CD4+ T cell pathogenicity.
Neutrophil depletion during sensitization led to a marked increase in Gata3+CD4+ T cells after multiple challenges, this increase was not apparent in lung-draining LNs after a single sensitization or after 2 sensitizations.
Neutrophil depletion during sensitization also failed to affect production of cytokines by cells prepared from mediastinal LNs (mLNs) 4 days postsensitization.
Depletion of neutrophils during sensitization has a pronounced, but delayed, impact on the phenotype and function of allergen-specific CD4+ T cells.
Neutrophil depletion during the sensitization phase increased allergen-specific CD4+ T cell pathogenicity.
LPS/OVA treatment led to marked increases in Tgfb1 RNA (Figure 6A), which encodes TGF-β, a well-described regulatory cytokine with many activities, including the ability to promote the differentiation of Tregs.
TGF-β protein also increased postsensitization, and was delayed compared with Tgfb1 RNA.
These increases occurred in parallel with accumulating neutrophils in the airway
Administration of the neutrophil-depleting anti-Ly6G antibody markedly reduced airway levels of TGF-β compared with animals that received no antibody or the IC antibody, thus confirming that neutrophils are required for the increased production of TGF-β during LPS-mediated allergic sensitization.
Neutrophil depletion had little effect on production of the proinflammatory cytokines IL-1α, IL-1β, TNF-α, or IL-6
This chemokine promoted the release of TGF-β into the airway; CXCL1 was as effective in this regard as 100 ng LPS.
ASP/OVA-mediated allergic sensitization, which does not trigger acute neutrophilia or lead to immunosuppression of established inflammation, failed to elicit TGF-β production.
Infiltrating neutrophils are both necessary and sufficient to induce TGF-β production and suggest that this neutrophil/TGF-β axis drives immunosuppressive responses to multiple allergen challenges.
Blocking TGF-β with a neutralizing antibody (clone 1D11) prior to LPS/OVA-mediated allergic sensitization led to much greater eosinophilia after a single OVA challenge and after multiple challenges.
TGF-β produced during LPS-mediated allergic sensitization through the airway is required for immunoregulatory responses to allergen challenge.
Neutrophil depletion during allergic sensitization again increased allergic responses to multiple subsequent allergen challenges; however, the addition of rmTGF-β during sensitization reversed this effect and restored immunosuppression.
Multiple daily OVA challenges were required for this effect, as the addition of rmTGF-β to neutrophil-depleted mice did not significantly affect responses to a single OVA challenge.
Discussion
Airway neutrophils can affect multiple features of asthma, including mucus hypersecretion and AHR.
Most known neutrophil functions are associated with asthma exacerbations, not with shaping the initiation of immune responses.
Neutrophils recruited to the airways during allergic sensitization may drive regulatory activities that function to suppress disease progression.
The nature and longevity of immune responses to inhaled OVA are remarkably sensitive to the amounts of LPS used as the adjuvant for allergen sensitization, with higher doses promoting both Th17 responses and shorter lived eosinophilic inflammation, compared with lower doses
Asthma prevalence is inversely associated with household levels of endotoxin; the current results suggest that the protective features of endotoxin might stem, at least in part, from its ability to recruit neutrophils to the airway during LPS-mediated allergic sensitization.
This recruitment, which does not occur during protease-mediated sensitization, results in increased airway levels of TGF-β that in turn suppress the pathogenicity of allergen-specific Th2 cells.
Blockade of neutrophil recruitment during sensitization unleashes the pathogenic potential of allergen-specific CD4+ T cells, as evidenced by their heightened ability to drive allergen-specific, eosinophilic inflammation upon their adoptive transfer into naive recipients.
Endotoxin present in house dust can also elicit neutrophil-dependent regulatory responses that limit the longevity of responses to sustained allergen challenge and may help explain why some epidemiologic studies find a negative association between household levels of endotoxin and asthma prevalence, whereas other studies find a positive association between these 2 parameters.
Neutrophil depletion augments allergic inflammation of the airway in a different model of asthma
Neutrophil depletion enhanced the actions of ILC2s, which were the major cellular source of IL-13
Highers amounts of ozone-induced IL-5 and IL-13 is produced by ILC2s from BALB/c mice than do their C57BL/6 counterparts.
The increased eosinophilia was not associated with increased amounts of IL-5 or the eosinophil chemoattractants, CCL11 and CCL24
Neutrophil-dependent changes in cytokine receptor expression and differential production of other secreted molecules can led to increased eosinophilia, this includes type 2 cytokines which were affected but not at the time points studied here.
TGF-β can promote the development of Tregs or together with the proinflammatory cytokines IL-1 and IL-6 to drive the differentiation of Th17 cells.
TGF-β is reported to drive ILC2 expansion, which might be anticipated to exacerbate ILC2-dependent allergic airway inflammation which suggest ILC2s are not driving the asthma-like phenotype in the current model.
Neutrophil depletion during sensitization was associated with significantly increased AHR,This contrast suggest different mechanisms of actions in the 2 studies.
Depleting neutrophils during allergic sensitization did not affect production of type 2 cytokines in lung-draining LNs shortly after sensitization or eosinophilic responses to a single allergen challenge whereas antibody-mediated blockade of TGF-β prior to sensitization was sufficient to increase eosinophilic responses to a subsequent single allergen challenge.
Recruitment of neutrophils to the airway likely acts to fine-tune levels of TGF-β, which in turn modulates the pathogenic potential of T cells. This regulatory mechanism might have evolved to limit tissue damage associated with type 2 immune responses to relatively harmless antigens, including allergens.
Indiscriminate blockade of neutrophil function might not always be helpful in the context of allergic asthma.
Methods
C57BL/6J, B6.Cg-Tg(TcraTcrb)425Cbn/J (OT-II), and B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) male mice were used in experiments at between 6 and 12 weeks of age. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the NIEHS, Research Triangle Park, North Carolina, USA.
Mice were sensitized with 50 μg LPS-free OVA with either 1 or 100 ng LPS from E. coli 0111:B4, 20 μg proteases from ASP, or 20 μL of HDEs. All o.p. instillations were in a total volume of 50 μL with sterile PBS as the diluent. Neutrophils were depleted 24 and 6 hours prior to sensitization by i.p. injections of 150 μg anti-Ly6G Ab (clone 1A8) or anti–Gr-1 Ab (anti-Ly6C/Ly6G; clone RB6-8C5). Where indicated, TGF-β1 was neutralized by 2 i.p. injections of 100 μg anti–TGF-β1 Ab (clone 1D11.16.8), given 24 and 6 hours prior to sensitization, with mouse IgG1 Ab (clone MOPC-21)
Previously sensitized mice were exposed 7 days after the second sensitization to an aerosol of 1% OVA for 1 hour on a single occasion or for 30 minutes on multiple consecutive days. BAL fluid was collected using 1 mL sterile PBS at the indicated times postsensitization for analysis of cellular inflammation and cytokines, whereas airway inflammation and AHR were assessed 48 hours after a single challenge or 24 hours after the last of several daily OVA challenges.
CD4+CD25+ Tregs were sorted from the lungs of C57BL/6 donor mice and suspended in suspended in complete RPMI media containing RPMI 1640, 10% fetal bovine serum, 0.1% 2-mercaptoethanol, 10 mM HEPES, and 100 U/mL penicillin/streptomycin.
Proliferative indices for CD4+ T cells from each experimental condition were generated by summing the percentage of cells corresponding to each division multiplied by that division number, thus, (% of undivided cells × 0) + (% of cells that underwent 1 division × 1) + (% of cells that underwent 2 divisions × 2), and so forth up to 6 divisions.
OVA-specific CD4+ T cells were prepared from spleens and LNs of OT-II mice or CD45.1 X OT-II mice. Lungswere enzymatically digested, then pressed through a 70 μm strainer, and the leukocytes were enriched using a histopaque gradient. CD4+ T cells were then purified from leukocytes by negative selection using biotin-labeled antibodies.
Enriched CD4+ T cells prepared from spleens and LNs of OT-II transgenic mice were transferred by retroorbital vein injection into recipient mice. These animals were then sensitized by o.p. instillation of OVA with a test adjuvant. Four days later, mLNs were excised, minced, and pressed through a 70 μm strainer, and 1 × 106 cells were cultured in 200 μL cRPMI media containing 10 μg/mL OVA. Culture supernatants were analyzed for cytokines.
Concentrations of TGF-β protein in whole lung lavage fluid were determined using commercial ELISA kits specific for activated mouse TGF-β.
Concentrations of IL-4, IL-5, IL-10, and IL-17 in BAL fluid or the supernatant of cultured regional LNs were measured as described previously, using a multiplexed fluorescent bead-based immunoassay.
Tgfb1 RNA expression was performed using a primer set from PrimerBank (ID 6755774c2), forward primer 5′-GAGCCCGAAGCGGACTACTA-3′ and reverse primer 5′-TGGTTTTCTCATAGATGGCGTTG-3′. Values were normalized to expression of the housekeeping gene 18s, forward primer 5′-CGGCTACCACATCCAAGGAA-3′ and reverse primer 5′-GCTGGAATTACCGCGGCT-3′.
Lymphocytes were enriched, diluted to μL, and incubated with a nonspecific binding blocking reagent cocktail of anti-mouse CD16/CD32 (2.4G2), normal mouse, and normal rat serum
Evaluations of AHR were performed using the FlexiVent mechanical ventilator system and assessing total respiratory system resistance after delivery of aerosolized methacholine.
Statistical calculations were performed using GraphPad Prism 7. Data are shown as mean ± SEM. Differences between groups were identified by Kruskal-Wallis 1-way ANOVA with Dunn’s multiple-comparison test, 1-way ANOVA with Dunnett’s or Holm-Šídák multiple-comparison tests, or 2-way ANOVA with Tukey’s multiple-comparison test. Individual comparisons between groups were confirmed by 2-tailed Student’s t test or the Mann-Whitney U test, and a P value of less than 0.05 was considered statistically significant.