Targeting IL-1β and IL-17A driven inflammation during influenza-induced exacerbations of chronic lung inflammation.

For patients with chronic lung diseases, such as chronic obstructive pulmonary disease (COPD), exacerbations are life-threatening events causing acute respiratory distress that can even lead to hospitalization and death. Although a great deal of effort has been put into research of exacerbations and potential treatment options, the exact underlying mechanisms are yet to be deciphered and no therapy that effectively targets the excessive inflammation is available. In this study, we report that interleukin-1β (IL-1β) and interleukin-17A (IL-17A) are key mediators of neutrophilic inflammation in influenza-induced exacerbations of chronic lung inflammation. Using a mouse model of disease, our data shows a role for IL-1β in mediating lung dysfunction, and in driving neutrophilic inflammation during the whole phase of viral infection. We further report a role for IL-17A as a mediator of IL-1β induced neutrophilia at early time points during influenza-induced exacerbations. Blocking of IL-17A or IL-1 resulted in a significant abrogation of neutrophil recruitment to the airways in the initial phase of infection or at the peak of viral replication, respectively. Therefore, IL-17A and IL-1β are potential targets for therapeutic treatment of viral exacerbations of chronic lung inflammation

Microbiota Promotes Chronic Pulmonary Inflammation by Enhancing IL-17A and Autoantibodies

RATIONALE Changes in the pulmonary microbiota are associated with progressive respiratory diseases including chronic obstructive pulmonary disease. Whether there is a causal relationship between these changes and disease progression remains unknown. OBJECTIVE To investigate the link between an altered microbiota and disease, we utilized a model of chronic lung inflammation in specific pathogen free (SPF) mice and mice depleted of microbiota by antibiotic treatment or devoid of a microbiota (axenic). METHODS Mice were challenged with LPS/elastase intranasally over 4 weeks, resulting in a chronically inflamed and damaged lung. The ensuing cellular infiltration, histological damage and decline in lung function were quantified. MEASUREMENTS AND MAIN RESULTS Similar to human disease, the composition of the pulmonary microbiota was altered in disease animals. We found that the microbiota richness and diversity were decreased in LPS/Elastase-treated mice, with an increased representation of the genera Pseudomonas, Lactobacillus and a reduction in Prevotella. Moreover, the microbiota was implicated in disease development as mice depleted of microbiota exhibited an improvement in lung function, reduction in airway inflammation, decrease in lymphoid neogenesis and auto-reactive antibody responses. The absence of microbial cues also markedly decreased the production of IL-17A, whilst intranasal transfer of fluid enriched with the pulmonary microbiota isolated from diseased mice enhanced IL-17A production in the lungs of antibiotic treated or axenic recipients. Finally, in mice harboring a microbiota, neutralizing IL-17A dampened inflammation and restored lung function. CONCLUSIONS Collectively, our data indicate that host-microbial cross-talk promotes inflammation and could underlie the chronicity of inflammatory lung diseases

IL-17A mediated neutrophilic inflammation during the initial phase of influenza-induced exacerbation.

<p>(A) BALB/c mice were treated with anti-IL-17A (α-IL-17A) or isotype control antibody one day prior and two days after infection with influenza virus (day 1–5 post infection) or PBS challenge (day 0). (B) Viral load was measured by quantitative real-time PCR and normalized to GAPDH. (C) The proportion of neutrophils in airways and lung were assessed by flow cytometry. Two representative FACS plots of airway neutrophilia at day 1 after infection are shown as well as the accumulated data. FACS plots show Ly-6C versus Ly-6G expression of CD11c⁻ CD11b⁺ pre-gated cells and indicate the frequency of neutrophils of all live cells. Data are representative of two independent experiments (n = 4–5), error bars indicate s.e.m; i.n. (intranasal); p.i. (post infection); ctrl (control).</p

Neutrophilic inflammation during influenza-induced exacerbation of COPD is mediated by IL-1β and IL-17A.

<p>(A) In the initial phase of viral replication, at 24 h following the infection, IL-1β-induced IL-17A caused the recruitment of neutrophils to the airways and lung. (B) At the peak of viral replication, day 5 post infection, neutrophilia became independent of IL-17A, but was still mediated by IL-1β. Blocking of IL-17A or IL-1β abrogated neutrophilic inflammation in the early phase of the infection or at the peak of viral replication, respectively.</p

Influenza infection induced exacerbation of established disease in LPS/elastase exposed mice.

<p>(A) Experimental protocol of influenza-induced exacerbation of LPS/elastase exposed mice. Control mice (indicated as day 0 in the following) were pre-exposed to LPS/elastase as were their infected counterparts, but challenged only with PBS instead of influenza virus. (B) Viral load of whole lung including airways and trachea was determined by quantitative real-time PCR at the indicated time points post infection or in non-infected mice (day 0) as control, respectively. Expression of influenza matrix protein was normalized to GAPDH. (C) Airway and tissue resistance was assessed by invasive plethysmography. (D) Absolute number of cells in the airways and lung was determined, and (E) the proportion of neutrophils and (F) inflammatory monocytes recruited to airways and lungs upon infection (day 3–7) or PBS challenge (day 0) was analyzed by flow cytometry. (G) Expression of IL-6 and TNFα was assessed by real-time PCR and normalized to GAPDH. (B)–(G) Experiments were performed in BALB/c mice and results are representative of at least two independent experiments (n = 4–5). Error bars represent s.e.m.; i.n. (intranasal).</p

Treatment with Anakinra reduced neutrophil recruitment to the airways at the peak of viral-induced inflammation.

<p>(A) BALB/c mice received Anakinra or PBS twice every day, starting two days prior to the viral infection and until mice were sacrificed. (B) Viral load was determined by quantitative real-time PCR and normalized to GAPDH. (C) Proportion of neutrophils recruited to the airways following influenza infection (day 1–5) or PBS challenge (day 0) was assessed by flow cytometry. Two representative FACS plots of each group at day 5 after infection are shown as well as the plotted data. FACS plots show Ly-6C versus Ly-6G expression of CD11c⁻ CD11b⁺ pre-gated cells and indicate the frequency of neutrophils of all live cells. Data are representative of two independent experiments (n = 4–5), error bars indicate s.e.m; i.n. (intranasal); p.i. (post infection); ctrl (control).</p

Model of chronic lung inflammation in BALB/c and C57BL/6 mice.

<p>BALB/c or C57BL/6 mice were exposed to LPS/elastase (L/E) or PBS once per week for four consecutive weeks as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098440#pone-0098440-g001" target="_blank">Figure 1A</a>. Disease severity was determined one week after the last LPS/elastase challenge. (B) Histological sections of lungs were stained with hematoxylin and eosin (H&E). (C) Destructive index (DI) and mean linear intercept (Lm) were scored from histological sections. (D) Cellular influx into airways was assessed by differential cell counts. All data are representative of at least two independent experiments (n = 3–5), error bars indicate standard error of the mean (s.e.m).</p

IL-1β mediated airway resistance, neutrophilic inflammation and IL-17A expression during influenza-induced exacerbations of COPD.

<p>Exacerbation of COPD in C57BL/6 mice was induced as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098440#pone-0098440-g002" target="_blank">Figure 2A</a>. (A) IL-1β protein in whole lung including airways and trachea following influenza infection (day 1–5) or PBS challenge (day 0) was assessed by ELISA. (B) Viral load in whole lung and trachea of wild type or IL-1β deficient animals was determined by quantitative real-time PCR and normalized to GAPDH. (C) Airway and tissue resistance was measured by invasive plethysmography at indicated time points after infection. (D) The proportion of neutrophils in the airways and lung was determined by flow cytometry. Data are pooled from two independent experiments (n = 4–5). (E) Expression of IL-6, TNFα, and (F) IL-17A was assessed by quantitative real-time PCR and normalized to GAPDH. (G) Proportion of IL-17A positive CD4⁺ T cells or γδ T cells was determined by flow cytometry after restimulation <i>in vitro</i>. All data are representative of at least two independent experiments (n = 4–5) and mean ± s.e.m. is shown.</p

Sustained Release of IL-2 Using an Injectable Hydrogel Prevents Autoimmune Diabetes

ABSTRACT Interleukin 2 (IL-2) is a promising therapy for autoimmune type 1 diabetes (T1D), but the short half-life in vivo (less than 6 minutes) limits effective tissue exposure to IL-2. Tissue exposure is required for the tolerogenic effects of IL-2. We have developed an injectable hydrogel that incorporates heparin polymers to enable the sustained release of IL-2. This platform uses clinical grade and commercially available materials, including collagen, hyaluronan, and heparin, to deliver IL-2 by slowly degrading and releasing IL-2 over a two-week period in vivo . We find that heparin potentiates the activity of IL-2 and IL-2-mediated expansion of Foxp3+ regulatory T cell (Treg). Hydrogel-mediated IL-2 release showed a reduction of CD4+ and CD8+ T cells and an increase of FoxP3+ Treg in the lymph nodes of injected mice. Moreover, in the Non-Obese Diabetic (NOD) mouse model of T1D once-weekly administration of IL-2 hydrogels prevented diabetes onset as efficiently as 3x weekly repeated injections of soluble IL-2. Together these data suggest that heparin-containing hydrogels may have benefit in delivering low-dose IL-2 and promoting immune tolerance in autoimmune diabetes