The sickle cell mouse lung: proinflammatory and primed for allergic inflammation

Abstract

Comorbid asthma in sickle cell disease (SCD) confers higher rates of vaso-occlusive pain and mortality, yet the physiological link between these two distinct diseases remains puzzling. We used a mouse model of SCD to study pulmonary immunology and physiology before and after the induction of allergic airway disease (AAD). SCD mice were sensitized with ovalbumin (OVA) and aluminum hydroxide by the intraperitoneal route followed by daily, nose-only OVA-aerosol challenge to induce AAD. The lungs of naive SCD mice showed signs of inflammatory and immune processes: (1) histologic and cytochemical evidence of airway inflammation compared with naive wild-type mice; (2) bronchoalveolar lavage (BAL) fluid contained increased total lymphocytes, %CD8+ T cells, granulocyte-colony stimulating factor, interleukin 5 (IL-5), IL-7, and chemokine (C-X-C motif) ligand (CXCL)1; and (3) lung tissue and hilar lymph node (HLN) had increased CD4+, CD8+, and regulatory T (Treg) cells. Furthermore, SCD mice at AAD demonstrated significant changes compared with the naive state: (1) BAL fluid with increased %CD4+ T cells and Treg cells, lower %CD8+ T cells, and decreased interferon gamma, CXCL10, chemokine (C-C motif) ligand 2, and IL-17; (2) serum with increased OVA-specific immunoglobulin E, IL-6, and IL-13, and decreased IL-1α and CXCL10; (3) no increase in Treg cells in the lung tissue or HLN; and (4) hyporesponsiveness to methacholine challenge. In conclusion, SCD mice have an altered immunologic pulmonary milieu and physiological responsiveness. These findings suggest that the clinical phenotype of AAD in SCD mice differs from that of wild-type mice and that individuals with SCD may also have a unique, divergent phenotype perhaps amenable to a different therapeutic approach.

Figure 1. OVA-Induced AAD (asthma) Model

To investigate the mechanisms and therapeutic targets of allergic asthma, we utilize a well characterized ovalbumin (OVA)-induced allergic airway disease (AAD) model. Following a three week period of OVA sensitization (one i.p. per week), mice are exposed to nose-only OVA-aerosol challenge for 1 hour per day for 3 days. (i.p. = intraperitoneal, SAC = sacrifice)

Figure 2. Sickle AAD mice have increased lung inflammation and mucus

Images are 20x magnification. Short arrows denote PAS+ stain indicative of mucus, whereas long arrows represent the peri-vascular/peri-bronchial inflammation. A= naïve C57, B = AAD C57, C= naïve hemizygous, D = AAD hemizygous, E = naïve SCD, F = AAD SCD. aw=airway. bv=blood vessel.

Figure 3. AAD induces shifts in BAL leukocyte counts and differentials in SCD mice but no difference in protein

Bronchoalveolar lavage (BAL) fluid was assessed for total leukocyte count (A), differentials (B, C) and protein concentration (D) from naïve C57 (n=6), hemizygous (n=6) and SCD (n=5) mice and compared to BAL from C57 (n=4), hemizygous (n=10) and SCD (n=7) mice after three weekly i.p. OVA-alum sensitizations followed by 3 days of OVA aerosolization. Boxes represent 25-75th percentiles. Whiskers represent minimum and maximum values. Medians are represented by a black or whitehorizontal line within boxes. Significant differences were determined by one-way ANOVA with multiple post-hoc pair-wise comparisons between groups using a student’s t-test. Differences between the naïve and AAD state of an individual mouse group is denoted by an asterisk (*). Differences between SCD mice and both C57 and hemizygous mice is denoted by two asterisks (**).

Figure 4. BAL T cell sub-populations are altered at baseline and at AAD in SCD mice

Lymphocyte flow cytometric analysis of BAL cells from C57, hemizygous, and SCD at baseline (n = 6, 6, and 5, respectively) and at AAD (n = 4, 10, and 7, respectively). CD4+ T cells are CD3+CD4+ lymphocytes. CD8+ T cells are CD3+CD8+ lymphocytes. Treg cells are CD3+CD4+CD25+Foxp3+ lymphocytes. Bars represent mean levels +/− SEM. Significant differences were determined by one-way ANOVA with multiple post-hoc, pair-wise comparisons between groups using a students’s t-test. Differences between the naïve and AAD state of an individual mouse group are denoted by an asterisk (*). Differences between C57 controls and SCD mice at AAD are denoted by two asterisks (**). Differences between C57 controls and both hemizygous and SCD mice in the naïve state or at AAD are denoted by three asterisks (***).

Figure 5. Naïve SCD mice have increased BAL G-CSF, IL-5, IL-7, and CXCL1

BAL was assessed from naïve C57 (n=6), hemizygous (n=6), and SCD (n=5) mice for cytokine and chemokine concentration via Luminex bead assay. Bars represent mean levels +/− SEM. Significant differences were determined by one-way ANOVA with multiple post-hoc, pair-wise comparisons between groups using a student’s t-test. Differences between SCD mice and both C57 and hemizygous mice are denoted by two asterisks (**).

Figure 6. AAD SCD mice have lower BAL concentrations of IFNγ, IL-17, CXCL10, and CCL2

BAL was assessed after induction of AAD from C57 (n=4), hemizygous (n=10), and SCD (n=7) mice for cytokine and chemokine concentration via Luminex bead assay. Bars represent mean levels +/− SEM. Significant differences were determined by one-way ANOVA with multiple post-hoc, pair-wise comparisons between groups using a student’s t-test. Differences between C57 mice and both hemizygous and SCD mice are denoted by three asterisks (***).

Figure 7. Serum OVA-specific IgE is 10-fold higher in SCD mice at AAD

Serum OVA-specific IgE was measured using a capture ELISA. C57 (n=9), hemizygous (n=9) and SCD (n=10) mice were analyzed after induction of AAD. Bars represent mean levels +/− SEM. Significant differences were determined by one-way ANOVA with multiple post-hoc, pair-wise comparisons between groups using a student’s t-test. Differences between SCD and C57 mice are denoted by two asterisks (**).

Figure 8. AAD induces an altered serum cytokine profile in SCD mice

Serum was assessed for AAD C57 (n=8), hemizygous (n=8), and SCD (n=8) mice for cytokine and chemokine concentration via Luminex bead assay. Bars represent mean levels +/− SEM. Significant differences were determined by one-way ANOVA with multiple post-hoc, pair-wise comparisons between groups using a student’s t-test. Differences between SCD mice and both C57 and hemizygous mice are denoted by two asterisks (**). Differences between C57 mice and both hemizygous and SCD mice are denoted by three asterisks (***).

Figure 9. Lung T cell sub-populations are increased in the naïve state but not at AAD in SCD mice

Lymphocyte flow cytometric analysis of lung mononuclear cells from C57, hemizygous, and SCD mice at baseline (n = 6, 6, and 4, respectively) and at AAD (n = 4, 10, and 7, respectively) was performed. CD4+ T cells are CD3+CD4+ lymphocytes. CD8+ T cells are CD3+CD8+ lymphocytes. Treg cells are CD3+CD4+CD25+Foxp3+ lymphocytes. Bars represent mean levels +/− SEM. Significant differences were determined by one-way ANOVA with multiple post-hoc, pair-wise comparisons between groups using a student’s t-test. Differences between the naïve and AAD state of an individual mouse group is denoted by an asterisk (*). Differences between SCD mice and C57 control mice at the naïve stage is denoted by two asterisks (**).

Figure 10. HLN lymphocyte sub-populations are altered in the naïve state and at AAD in SCD mice

Lymphocyte flow cytometric analysis of hilar lymph node (HLN) mononuclear cells from C57, hemizygous, and SCD mice at baseline (n = 6, 6, and 4, respectively) and at AAD (n = 9, 9, and 10, respectively) was performed. CD4+ T cells are CD3+CD4+ lymphocytes. CD8+ T cells are CD3+CD8+ lymphocytes. Treg cells are CD3+CD4+CD25+Foxp3+ lymphocytes. Bars represent mean levels +/− SEM. Significant differences were determined by one-way ANOVA with multiple post-hoc, pair-wise comparisons between groups using a student’s t-test. Differences between the naïve and AAD state of an individual mouse group is denoted by an asterisk (*). Differences between SCD and hemizygous mice compared to C57 mice at the naïve stage is denoted by two asterisks (**). Differences between C57 mice and SCD mice at AAD are denoted by three asterisks (***).

Figure 11. SCD and hemizygous mice are hyporesponsive to methacholine challenge at AAD

Airway hyperesponsiveness was assessed at AAD following 3 OVA i.p. sensitizations and 3 days of OVA aerosol challenge. Sensitivity to methacholine challenge was compared between SCD (n=7, closed triangles), hemizygous (n=7, open boxes), and C57 control (n=6, closed circles) mice. Increasing concentrations of methacholine are denoted on the x-axis. Area under the curve (AUC) was computed for each mouse and groups compared using one-way ANOVA and Tukey post-hoc test. Data represent mean ± SEM levels of Penh responses. The overall effect of genotype on methacholine response is significant (p < 0.01). p < 0.01 for C57 versus SCD. p = 0.06 for C57 versus Hemizygous.