Dissecting asthma pathogenesis through study of patterns of cellular traffic indicative of molecular switches operative in inflammation

Abstract

Background: Inflammation and degeneration are the two edged swords that impale a pulmonary system with the maladies like asthma and idiopathic pulmonary fibrosis. To explore critical role players that orchestrate the etiology and pathogenesis of these diseases, we used various lung disease models in mice in specific genetic knockout templates.

Materials and methods: Acute and chronic allergic asthma and idiopathic pulmonary fibrosis model in mouse was developed in various genetic knockout templates namely α4^{Δ/ Δ}(α41-/-), β2-/-, and α4-/- β2 mice, and the following parameters were measured to assess development of composite asthma phenotype- (i) airway hyperresponsiveness to methacholine by measuring lung resistance and compliance by invasive and P_enh by non-invasive plethysmography as well as lung resistance and compliance using invasive plethysmography, (ii) in situ inflammation status in lung parenchyma and lung interstitium and also resultant airway remodelling measured by histochemical staining namely Masson's Trichrome staining and Hematoxylin&Eosin staining, (iii) formation of metaplastic goblet cells around lung airways by Alcian blue dye, (iv) measurement of Th1 and Th2 cytokines in serum and bronchoalveolar lavage fluid (BALf), (v) serum allergen-specific IgE. Specifically, ovalbumin-induced acute allergic asthma model in mice was generated in WT (wildtype) and KO (knockout) models and readouts of the composite asthma phenotype viz. airway hypersensitivity, serum OVA-specific IgE and IgG, Th2 cytokine in bronchoalveolar lavage fluid (BALf) and lymphocyte cell subsets viz. T, B cells, monocytes, macrophages, basophils, mast cells and eosinophils (by FACS and morphometry in H&E stained cell smears) were assessed in addition to lung and lymph node histology.

Results: We noticed a pattern of cellular traffic between bone marrow (BM)→ peripheral blood (PB) → lung parenchyma (LP) → (BALf) in terms of cellular recruitment of key cell sub-types critical for onset and development of the diseases which is different for maintenance and exacerbations in chronic cyclically occurring asthma that leads to airway remodelling. While inflammation is the central theme of this particular disease, degeneration and shift in cellular profile, subtly modifying the clinical nature of the disease were also noted. In addition we recorded the pattern of cell movement between the secondary lymphoid organs namely, the cervical, axillary, ingunal, and mesenteric lymph nodes vis-à-vis spleen and their sites of poiesis BM, PB and lung tissue. While mechanistic role is the chief domain of the integrins (α4 i.e. VLA-4 or α4β1, VCAM-1; β2 i.e. CD18 or ICAM-1).

Concluding remarks: The present paper thoroughly compares and formulates the pattern of cellular traffic among the three nodes of information throughput in allergic asthma immunobiology, namely, primary lymphoid organs (PLO), secondary lymphoid organs (SLO), and tissue spaces and cells where inflammation and degeneration is occurring within the purview of the disease pathophysiological onset and ancillary signals in the above models and reports some interesting findings with respect to adult lung stem cell niches and its resident progenitors and their role in pathogenesis and disease amelioration.

Keywords: Bone marrow; GMP; Mesenchymal stem cell; Platelet rich plasma; Serum free medium.

Figure 1.

Study protocol for transplantation for hematopoietic reconstitution probing mobilization and homing.

Figure 2.

Study design to generate acute allergic asthma phenotype in mice.

Figure 3.

Distribution of alpha4+ cells in primary and secondary lymphoid organs: donor neonatally ablated Mx.cre alpha4-/- vs.

Figure 4.

Donor BM CFU-C assessed before the transplant.

Figure 5.

Progenitors in different tissues of the three genotype groups.

Fig 6A.

Cellularity in thymus

Figure 6B.

Distribution of different development stages of B and T cells in BM, PB and Spleen. Graph of cell markers in BM, PB and Spleen, CD3+

Figure 6C.

Distribution of different development stages of T cell subsets in BM, PB and Spleen; Graph of cell markers in BM, PB and Spleen, CD4+, CD8+

Figure 6D.

Distribution of different developmental stages of T cells in BM, PB and spleen. Graph of cell markers in BM, PB and Spleen, CD4+CD25+ Treg.

Figure 6E.

Distribution of different developmental stages of T cells in BM, PB and spleen. Graph of cell markers in BM, PB and Spleen, Progenitor B cells

Figure 6F.

Distribution of different developmental stages of T cells in BM, PB and spleen. Graph of cell markers in BM, PB and Spleen, Pre B cells B220+CD34-

Figure 6G.

Distribution of different developmental stages of T cells in BM, PB and spleen. Graph of cell markers in BM, PB and Spleen, Pro B cells – Early B cells

Figure 6H.

Distribution of different developmental stages of T cells in BM, PB and spleen. Graph of cell markers in BM, PB and Spleen, Mature B cells (B220+IgM+)

Figure 7.

Mediators of differentiation pathways from stem cells.

Figure 8.

Ramifications of myriad factors operative in inflammation in complex network.

Figure 9.

Schematic representation of the key inflammasomes and their pathways.

Figure 10.

Summary findings and conclusion - Tentative scheme of cellular traffic.