Loss of alveolar capillary membrane integrity leads to Activation of Th2 cells

The biological plasubility of this KER is high as there is a mechanistic relationship between alveolar capillary membrane (ACM) injury (tissue damage), and the induction of a Th2 response (responsible for wound healing) (Gieseck et al., 2018; Wynn, 2004).

The empirical support for this KER is moderate. There is limited in vitro and in vivo evidence to support a direct relationship between these two KEs, with some inconsistencies with respect to the specific mediators in question (Ortiz et al., 1998; Piguet, 1989; Redente et al., 2014; Additional references can be found in Table 1).

In mice lacking both Tumor necrosis factor receptor 1 (TNF-R1) and receptor 2 (TNF-R2) or in wild-type (WT) mice treated with anti-TNFα, bleomycin-induced lung fibrosis is attenuated (Ortiz, 1998; Piguet, 1989). Persistent activation of TNF-α and IL-1β results in elevated secretion of pro-inflammatory cytokines that are tissue damaging. Overexpression of IL-1β induces acute lung injury and lung fibrosis in mice (Kolb, 2001). TNF-α and IL-1β are the therapeutic targets in IPF and asbestosis (Zhang et al., 1994). Overexpression of TNF-α induces spontaneous fibrosis in mouse lungs (Miyazaki et al., 1995). In cases of infestation with parasitic worm helminths, chronic injury activates a large immune response, resulting in secretion of pro-inflammatory mediators that can inflict cell and tissue damage. Effective treatment involves control of immune-response mediated damage (reviewed in Jackson et al., 2009).

Dose-Response Relationship:

There are some in vivo studies that provide dose-response evidence of this KER.

Li et al. (2017) studied the immunotoxic effects of the lung after arsenic exposure in an acute and a subchronic phase. Female C57BL/6 mice were exposed to 2.5, 5, and 10 mg/Kg sodium arsenite (NaAsO₂) via a single oral intragastric administration. After 24 h, bronchoalveolar lavage fluid (BALF) and lung tissue were collected. The induction of KE3 (Event 1498; ACM injury) was observed as an increase in the total protein levels in BALF, malondialdehyde content, and Nuclear factor erythroid 2-related factor 2 (Nrf2) protein expression in the lung. The induction of KE4 (Event 1499; Th2 activation) was determined as an increase in the levels of Gata3, IL-4, Forkhead box P3 (Foxp3), and IL-10 levels, as well as a decrease in the levels of T-box transcription factor (T-bet), Interferon gamma (Ifn-γ), Retinoic acid-related orphan receptor gamma t (Ror-γt), and IL-23. All these changes were observed in a dose-dependent manner. After a subchronic exposure (100 mg/mL NaAsO₂ administered freely in drinking water for 2 months), an increase in the levels of Nuclear Factor Kappa B (NF-κB), p-38 (a mitogen-activated protein kinase), Phospho-c-Jun N-terminal kinase (p-JNK), Phospho-extracellular signal-regulated kinase (p-ERK) mRNA and an increase in the levels of IL-4, IL-23, IL-10, Ifn-γ, IL-1β was observed.

Chang et al. (2017) studied the role of NF-κB activation and Th1/Th2 imbalance in pulmonary inflammation induced by Nickel oxide (NiO) nanoparticles (NPs). Male Wistar rats were exposed to 0.015, 0.06, and 0.24 mg/Kg by intratracheal instillation twice a week for 6 weeks. At the highest dose, an increase of nitrative stress in rat lung tissue was observed. TNF-α, IL-2, IL-10, and Cytokine-induced neutrophil chemoattractant (CINC) increased in a dose-response manner. Activation of the NF-κB signalling pathway (NF-κB, Inhibitor of NF-κB kinase subunit alpha [IKK-α] and NF-κB-inducing kinase [NIK]) also increased in a dose-response manner. An increase in the levels of GATA3 and a decrease in T-bet was observed, indicating a Th1/T2 imbalance after exposure to NiO NPs. NiO NPs the nitrative stress and inflammatory response in lung tissue, related to NF-κB and Th1/Th2 imbalance.

Temporal Evidence:

In vivo studies have demonstrated that the loss of ACM integrity precedes the activation of Th type 2 cells.

It has been described that oxidant and antioxidant Th1/Th2 balances are essential in the inflammatory response and fibrosis. To evaluate whether transcription factor Nrf2 is a mediator, WT C57BL/6 mice, and Nrf2-deficient mice were administered with 5 mg/kg bleomycin intratracheally, and the inflammatory response, antioxidant response, and fibrosis were evaluated at different time points post-exposure. The loss of ACM integrity was observed at days 1 and 3, as well as a decrease in the induction of antioxidant genes. On day 7, an increase in the level of Th2 cytokine production (IL-4 and IL-13) was observed. The expression of GATA-3, a transcription factor that regulates the differentiation of Th1/Th2 cells, was also increased at this time point. These responses were more intense in Nrf2-deficient mice. Moreover, bleomycin administration increased the recruitment of cells at day 1 and 3, and fibrosis were observed after 28 days of treatment (Kikuchi et al., 2010).

Haczku et al. (2006) observed in mice sensitized and challenged with Aspergillus fumigatus extract (intraperitoneal injection at day 0 and day 14, intranasal challenge at day 24) an increase in the expression of surfactant protein D (SP-D) mRNA and protein levels in lungs in a time-dependent manner after allergen challenge. Total BALF protein increased over time at 12 and 24 h; after that, protein content decreased. IL-4, IL-5, and IL-13 increased at 12 h and 24 h, but decreased at later time points. They found that allergen exposure increased the expression of SP-D and IL-4/IL-13 in a time-dependent manner.

Venosa et al. (2016) evaluated the presence of classically activated (M1) macrophages and alternatively activated (M2) macrophages in lung after the exposure to nitrogen mustard. Rats were exposed to nitrogen mustard at 0.125 mg/kg, intratracheally, and the inflammatory response was evaluated 1, 3, 7- and 28-days post-exposure. A decrease in the levels of resident macrophages and an accumulation of infiltrating M1 and M2 macrophages was observed. M1 macrophages were prominent after 1- and 3-days of exposure; meanwhile, M2 macrophages were more prominent after 28 days of exposure. M1 and M2 genes were also upregulated. These events were in a time-dependent manner. M2 macrophages in early time points phagocytized cellular debris and counterbalanced M1 cell activation. At later time points, they promoted matrix deposition, tissue remodeling, and fibrosis.

Caldwell et al. (2009) studied the inflammatory response after exotoxin pyocyanin exposure in WT and FVBN mice. Mice were exposed to pyocyanin 10 or 25 µg intranasally inoculated chronically into the lungs three times a week for intervals of 3, 6, and 12 weeks. At 25 µg, after 6 and 12 weeks, alveolar airspace destruction was observed (KE3 (Event 1498)). The KE4 event (Event 1499) decreased resident macrophages and increased CDT cells/ml in BALF, changed in a time-dependent manner with maximum levels after 12 weeks. STAT6 mediated the induction of Th2 cytokines by pyocyanin.

Exogenous delivery of TNF-α to mouse lungs with established fibrosis, reduced the fibrotic burden. Exogenous treatment with TNF-α slowed the M2 macrophage polarisation. TNF-α deficient mice showed prolonged pro-fibrotic response and M2 polarisation following bleomycin treatment (Redente et al., 2014).