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

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 α4Δ/ 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 Penh 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.


Introduction
Inflammation is meant to re-establish a shift in the body's homeostatic balance. Acute inflammation is the initial response to harmful stimuli and is achieved by the increased movement of plasma and leukocytes from blood into injured tissues concomitant with a cascade of biochemical events involving the systemic role of vascular and immune system and local role of other tissue specific cells within the injured tissue (ZF, 2007). Prolonged inflammation or chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process. This is characterized by concurrent active inflammation, tissue destruction, and attempts at repair and may not be typically characterized by the aforementioned classic signs of acute inflammation.
Instead, chronically inflamed tissue is characterized by the infiltration of mononuclear immune cells (monocytes, macrophages, lymphocytes, and plasma cells), tissue destruction, and attempts at healing, which include angiogenesis and fibrosis (Yang, 2006). Asthma and COPD although differ significantly in their underlying etiology, involve similar inflammatory changes in the respiratory tract. While the specific nature and the reversibility of these processes largely differ in each entity and disease stage, both are characterized by lung inflammation; though patients with asthma suffer largely from reversible airflow obstruction, whereas patients with COPD experience a continuous decline in lung function as disease progresses (Davidson et al., 2010). By 2020 India alone will account for 18% of the 8.4 million tobacco related deaths globally (Sharma P, 2009).
In China, COPD is one of the high frequency causes of death followed closely by Ischemic heart disease and cardiovascular disease (Broide et al., 1998).
Inflammation is therefore key to etiology of most respiratory disorders and while it is critical for the body's defence against infections and tissue damage, it has increasingly become clear that there is a fine balance between the beneficial effects of inflammation cascades and potential for tissue destruction in the long term. If they are not controlled or resolved, inflammation cascades lead to development of diseases such as chronic asthma, rheumatoid arthritis, psoriasis, multiple sclerosis and inflammatory bowel disease (Takizawa, 2007).
The specific characteristics of inflammatory response in each disease and site of inflammation may differ but recruitment and activation of inflammatory cells and changes in structural cells remain a universal feature. This is associated with increase in the expression of components of inflammatory cascade viz. cytokines, chemokines, growth factors, enzymes, receptors, adhesion molecules and other biochemical mediators.
The pathogenesis of allergic asthma involves the recruitment and activation of many inflammatory and structural cells, all of which release mediators that result in typical pathological changes of asthma. The chronic airway inflammation of asthma is unique in that the airway wall is infiltrated by T lymphocytes of the T-helper (Th) type 2 phenotype, eosinophils, macrophages/monocytes and mast cells. Accumulation of inflammatory cells in the lung and airways, epithelial desquamation, goblet cell hyperplasia, mucus hyper-secretion and thickening of submucosa resulting in bronchoconstriction and airway hyperresponsiveness are important features of asthma (Czarnobilska E, 2005;Murphy, 2010). Both cells from among the circulating leukocytes such as Th2 lymphocytes, mature plasma cells expressing IgE, eosinophils (Murphy, 2010) and neutrophils as well as local resident and structural cells constituting the 'respiratory membrane' (airway epithelial cells, fibroblasts, resident macrophages, bronchial smooth muscle cells, mast cells etc.) contribute to the pathogenesis of asthma (Henderson et al., 2002). Airway hyperresponsiveness of asthma is clinically associated with recurrent episodes of wheezing, breathlessness, chest tightness and coughing, particularly at night or in early morning. Furthermore, during exacerbations the features of "acute on chronic" inflammation have been observed. Chronic inflammation may also lead to the outlined structural changes often referred to as airway remodelling which often accounts for the irreversible component of airway obstruction observed in some patients with moderate to severe asthma and the declining lung function.
Inflammation in COPD is associated with an inflammatory infiltrate composed of eosinophils, macrophages, neutrophils, and CD8 + T lymphocytes in all lung compartments (Henderson et al., 2002) along with inflammatory mediators such as TNF-, IL-8 (interleukin-8), LTB4 (Leucotriene B4), ET-1 (Endothelin-1) and increased expression of several adhesion molecules such as ICAM-1 (Woodside and Vanderslice, 2008). The molecular mechanisms whereby inflammatory mediators are upregulated at exacerbation may be through activation of transcription factors such as nuclear factor (NF)-κB and activator protein-1 that increase transcription of proinflammatory genes (Erlandsen et al., 1993). Acute exacerbations have a direct effect on disease progression by accelerating loss of lung functional though the inflammatory response at exacerbation is variable and may depend in part on the etiologic agent (Poole, 2001;Troosters et al., 2010). Current therapies for COPD exacerbations are of limited effectiveness (Rennard et al., 2007).
Rational treatment depends on understanding the underlying disease process and there have been recent advances in understanding the cellular and molecular mechanisms that may be involved (Rennard et al., 2007 (Calverley et al., 2007). Targeting oxidative damage using antioxidants such as N-acetylcysteine as shown efficacy in chronic bronchitis (Erlandsen et al., 1993) but is relatively ineffective in established COPD (Holgate, 2007).
Targeting TNF-to ameliorate inflammation has also been disappointing (Cushley et al., 1983;Nakajima, 1994). The use of inhaled steroids combined with longacting β2 agonists to reduce exacerbation rates in more severe disease is now widely accepted, but their effects on mortality are still in doubt (Laberge et al., 1995) and presently there are no effective strategies beyond smoking cessation to slow disease progression in horizon (Schneider et al., 1999). These data suggest that even relatively modest immunomodulators such as inhaled corticosteroids might further impact on local immunity already damaged by chronic inflammation and remodelling, rendering individuals to some degree more vulnerable to significant infections (Chin, 1997). Key to effective COPD therapy is prevention of loss of alveolar smooth muscle elasticity which is irreversible by early diagnosis and more effective intervention which is currently virtually non-existent.
It is with an objective to identify targets that conventional therapy has obviously overlooked or underrated, that the pattern of cellular traffic is being studied under various patho-physiological situations. A number of genetic knockout models of mice were used and tissue specific (lung) inflammation under asthmatic (Th1-driven) condition was explored and immune cell traffic from their site of poiesis to their site of pathophysiological manifestation were studied (Henderson et al., 1997).
In our work with various genotype knockout models of mice, the data generated and interpreted from
poly(I)poly(C) injection. cremice were used as WT (wild type) and 4 ablated mice were simply called 4-/-. CD18-/-mice on a C57BL6 background were called 2-/-. In total the following number of animals were used in each group:    Cell number refers to that obtained from lavage of both lungs/mouse.

Lung parenchyma
Lung mincing and digestion was performed after lavage as described previously with 100u/ml collagenase for 1 hr at 37 0 C, and filtered through a 60# sieve (Sigma).
All numbers mentioned in this paper refer to cells obtained from one lung/mouse.

Lung histology
Lungs of other animals of same group were were fixed in 4% paraformaldehyde overnight at 4 0 C. The tissues were embedded in paraffin and cut into 5 m

OVA specific IgE and IgG1 in serum
Anti-mouse IgE (R35-72) and IgG1 (A85-1) from BD Biosciences, San Diego, CA were used for measuring OVA specific IgE and IgG1 (in serum previously frozen at -70 0 C) respectively by standard ELISA procedures as previously described.

Results
Rationale for the study  (Lee et al., 2003;Scott et al., 2003).   However, it is notable that CD8+cells were at very low levels in thymus and lower than controls, in contrast to levels in PB (w1.9:1).
Traffic between primary and secondary lymphoid tissue There was a tendency for preferential migration of  recruited from blood to tissue as shown in Table 2   As regards the secondary lymphoid organs and tissues, the first to be considered was CLN as it is anatomically proximal to the lung. Table 9A Table 7. Total count (Lymphoid and myeloid cells) in trachea. Number of cells in PLO and SLO and tissue were calculated as tC and DC by hemocytometric analysis in a standard Neubauer's Hemocytometer, DC was analyzed by double blind counting from H&E stained smears prepared in a cytospin (manufactured by Vision Scientific, South Korea, model Centurion Scientific C2 series) using Zeiss photo allotment and Axiostar plus software and by flow cytometry using BD flow cytometer (BD Accuri C6 cytometer) and analyzed by BD Accuri C6 software using monoclonal fluorochrome tagged antibody as mentioned in Materials & Methods.  Table 8. Total count (Lymphoid and myeloid cells) in spleen. Number of cells in PLO and SLO and tissue were calculated as tC and DC by hemocytometric analysis in a standard Neubauer's Hemocytometer, DC was analyzed by double blind counting from H&E stained smears prepared in a cytospin using Zeiss photo allotment and Axiostar plus software and by flow cytometry using BD flow cytometer and analyzed by BD Accuri C6 software using monoclonal fluorochrome tagged antibody as mentioned in Materials & Methods. At the outset, we must consider the significance of the donors that were chosen and the recipients' pretreatment before transplantation. It may be conclusively deduced from Fig.3 that some SLO are resistant to ablation of this integrin. The reason may be important metabolomic and transcriptomic pathways that disallows this resulting in the immunosecretome being readjusted and repositioned such that ablation of α4 is compensated. Fig.4 however, unambiguously excludes BMC from such non-permissiveness to ablation ensuring a 100% αa free reconstitution. As seen in previous publications, (Banerjee et al., 2009;Banerjee et al., 2008;Ray Banerjee, 2007;Scott et al., 2003), α4 has regulatory roles in mobilization, homing, and engraftment which explains their higher indicate their diverse pathways action as enumerated in (Banerjee et al., 2009;Ray Banerjee, 2007;Ulyanova et al., 2007). But what is interesting here is the complete separation of the cell traffic (direction and number) as evident from Tables 1-8   and that denotes their exact counterparts in mouse as they will be extrapolated to represent.
Mortality being low and repopulation of transplanted cells being 100% by 48 weeks, we can say safely that the transplanted BMC did their job well, that is they found the regions to home to and then they settled down there and all progenitors generated new clones and differentiated into the type of cells that the tissues needed.
In other words there was induction of differentiation in situ.
As for the rate limiting steps which are key in cellular traffic resulting in onset and development of the actual disease, α4 was found to be a critical regulatory factor for myeloid cellular traffic compared to lymphoid.
( Table 1) Overall, both integrins seem to have regulatory roles in lymphoid and nonlymphoid cell migration to blood.
The number of cells in BALf is the most striking and as described in (Banerjee et al., 2009;Banerjee et al., 2008;Ray Banerjee, 2011b;Ulyanova et al., 2007) that while α4 principally controls sensitization and signaling, β2 mainly control the migration from lung parenchyma to interstitium without which onset of the asthma immunopathology cannot occur. Table 2      therefore lymphoid cells as they migrate from blood to lung tissue and thence to the interstitial spaces where the actual gas exchange occurs and this step is almost exclusively controlled by the β2 integrin. α4 controls the lymphoid signaling while β2 controls myeloid migration. So these are the rate limiting cells from PLO to tissue. Table 3 reveals conclusively that although in α4-/-BM, synthesis and sequestration of T cells fall quite tremendously and post-OVA their number is amplified manifolds more than in WT. As mentioned in (Scott et al., 2003), this may either be due to a lack of "settling down" on the stroma of the PLO or a genuine increase in synthesis of these cells but it certainly means that there is possibly no impairment in the upstream IL-4-IL-4R and IL-5-induced recruitment from bone marrow in its absence. Data shown in Table 7D coupled with data presented in Fig.6A highlight that α4 is probably key to the Th1-Th2 balance.
In Table 5-7C, data shows that although IL-4/5 induced cell recruitment is probably still operative despite absence of β2-cells from these B cells, they are unable to cross the number threshold and therefore insufficient to mount a viable pathophysiological circuit. (Banerjee et al., 2008;Farrell, 2003;Lee et al., 2003;Ray Banerjee, 2007;Scott et al., 2003;Ulyanova et al., 2007;Weber et al., 1992).
There may be a role for epithelial progenitors, circulating and niche-dwelling (Gomperts et al., 2006) which is beyond the scope of this paper and is being currently investigated.

Summary findings & conclusion-Highlights
Observations 1. α-/-cells are slower to reconstitute 2. Increased hematopoiesis in all primary & secondary lymphoid organs in KO gr 3. Myeloid repopulation was similar in both groups 4. B cells, pro B cells, pre B cells, mature B cells significantly decreased in KO gr 5. Total T cells increased but activated T cells decreased in KO gr 6. Repopulation of thymus was significantly impaired in KO gr 7. CD+CD8+ was more in thymus and CD+ differentiation was skewed in KO gr 8. OVA-induced acute allergic asthma phenotype was impaired in all aspects 9. All PLNs show decreased mature B cells 10. Enhanced tendency of memory T cells (CD45RC-CD4+) to migrate to LNs in KO gr 11. Enhanced tendency of memory T cells (CD45RC+CD4+) to migrate to spleen & thymus 12. Functionality & homing impairment in KO gr 13. α-/-probably do not have a role to play during response to OVA per se, and neither to specifically T cell proliferation in response to the upstream cytokine signaling in response to OVA.

Salient findings and clinical implications
Various small molecules inhibitors for α4 integrin such as Tysabri (Roche) and integrin Beta2/CD18 antibodies products manufactured by Biolegend and Novus Biologicals and others which are specifically designed for modulation of the target molecule must take into account targeting strategies along with the timing of drug activation in a particular tissue (in pro drug formulation, through nano-vehicles for timed and tissue specific delivery. Positioning of PLO, SLO and inflamed tissue as nodes of cellular and information traffic and the network operative in specific temporal templates is crucial information for the success of such drugs. The role of α4 and β2 are very much cell specific and tissue specific, for example their role in bone marrow, blood, lung, interstitial tissue spaces, trachea, and lymph nodes are not uniform.
The "kicking in" is initiated possibly by α4 during initiation and "priming" of the B cells and T cells.

Conflict of interest
The author has declared that no conflict of interest exists.