The Relm family of proteins was originally identified in the lung and gastrointestinal tract and is strongly linked with the induction of Th2 immune responses and mucosal immunity, involving asthma, helminthic parasites, and inflammatory bowel disease (15
). Relm-α is a hallmark signature gene of murine alternatively activated macrophages (25
). However, epithelial cells, eosinophils, and adipose tissue may also express Relm-α (16
). Surprisingly, despite intensive research into Relm-α, its role in models of asthma remains unknown.
In this study, we demonstrate several key and unexpected results regarding the regulation and roles of Relm-α, using acute models of experimental asthma. First, we demonstrate the differential regulation of Relm-α by IL-13Rα1, and likely the Type I IL-4R, depending on the experimental asthma model. In particular, we demonstrate that IL-13Rα1 critically regulates Relm-α expression after Asp challenge. However, after OVA + alum–induced experimental asthma, Relm-α expression is regulated by both IL-13Rα1 and most likely the Type I IL-4R. Second, we demonstrate that baseline Relm-α expression is restricted to airway epithelial cells, whereas after the induction of experimental asthma, airway epithelial cells and macrophages express Relm-α, consistent with the presence of alternatively activated macrophages only after the induction of Th2-associated lung disease. Third, we demonstrate that Relm-α partly regulates IL-13–induced lung chemokine production. In particular, Relm-α–deficient mice display elevated concentrations of CCL24, CCL2, and CCL11 and decreased concentrations of CCL17 and CCL22. Notably, IL-13–challenged Retnlb−/− mice displayed significantly more CCL2 and less CCL22 than IL-13–challenged Retnla−/− mice. Finally, using two models of experimental asthma, we demonstrate that Relm-α does not have a marked role in the overall Th2 response in the lung, as assessed by the production of lung Th2 cytokine, chemokine, cellular recruitment, and mucus production.
Our results demonstrate that IL-13Rα1 differentially regulates Relm-α expression. After OVA + alum sensitization and consequent intranasal OVA challenge, Relm-α expression is predominantly IL-13Rα1–independent, whereas after mucosal sensitization and challenge (using Asp extract), the expression of Relm-α was IL-13Rα1–dependent. Whereas airway epithelial cells predominantly express Type II IL-4R, which mediates IL-4 and IL-13 signaling, infiltrating hematopoietic cells predominantly express Type I IL-4R (40
). Indeed, we demonstrate that after allergen challenge, Relm-α is expressed by both airway epithelial cells and macrophages. Therefore, the differential regulation and expression of Relm-α are likely driven by the marked differences between the OVA + alum and Asp models and may result from differential IL-4 versus IL-13 production (14
), as the ratio between IL-13 and IL-4 is substantially higher after Asp inoculation than after OVA inoculation (13
). Despite our finding that lung eosinophils express Relm-α mRNA and that gastrointestinal eosinophils express Relm-α protein, murine eosinophils did not express Relm-α protein after IL-13 challenge or allergen challenge. These results may indicate different roles for eosinophils or eosinophil-derived Relm-α in innate-immune gastrointestinal inflammatory settings compared with allergic airway inflammation.
Given the strong association between IL-13 and Relm-α induction and the ability of IL-13 to induce Relm-α directly, we hypothesized that Relm-α would regulate IL-13–induced lung responses. In particular, IL-13–treated Retnla−/−
mice displayed altered chemokine induction. CCL17 and CCL22 were decreased in Retnla−/−
mice, indicating a role for Relm-α in the induction of these chemokines. CCL17 and CCL22 are mainly implicated in the recruitment of Th2 T-cells (41
). Although we could not detect any alterations in IL-13–induced T-cell recruitment into the lung, in different settings, Relm-α may be able to modulate T-cell responses by governing their chemotactic signals. Moreover, CCL24 (a hallmark eosinophil chemokine) (42
) and CCL2 (which recruits monocytes and dendritic cells) (44
) were increased in IL-13–challenged Retnla−/−
mice, indicating a suppressive role for Relm-α. Collectively, these data suggest that the effect of Relm-α was predominantly attributable to the effects of Relm-α on airway structural cells (such as epithelial cells), as these are responsive to IL-13 in the induction of chemokine production (40
). Furthermore, given the structural similarities between Relm-α and Relm-β (15
), we were interested in determining whether Relm-regulated IL-13–induced lung chemokine production was Relm-α–specific. The regulatory effects of Relm-α and Relm-β on IL-13–induced chemokine production were similar, although Relm-β was more potent at increasing CCL24 and CCL2 and decreasing CCL17 and CCL22. Although the receptors for Relm-α and Relm-β remain unknown, the different potencies of Relm-α and Relm-β in regard to IL-13–induced chemokine production may partly attributable to their respective receptor expression or the induction of intracellular signaling.
Recent studies indicate a key role for Relm-α in helminth-induced Th2 responses () (18
). Indeed, Nippostrongylus
mice display significantly increased Th2 cytokine production (including IL-4, IL-5, and IL-13) 7 days after infection. Furthermore, Schistosoma mansoni
mice displayed increased lung pathology (increased size of egg-induced granulomas, and elevated fibrosis), which was associated with elevated Th2 cytokines and IgE production (23
). In fact, macrophage-derived Relm-α was shown to negatively regulate Th2 cytokine production from anti-CD3/anti-CD28–stimulated splenocytes. Collectively, these data suggest that Relm-α would also be a negative regulator of allergen-induced allergic airway inflammation. Nevertheless, in response to allergen challenge, using two distinct experimental asthma models, Retnla−/−
mice displayed similar Th2 cytokine production compared with WT mice. Furthermore, mucus production and chemokine induction were similar in allergen-exposed Retnla−/−
and WT mice.
COMPREHENSIVE SUMMARY COMPARING EFFECTS REGULATED BY RELM-α AND ARGINASE 1 IN EXPERIMENTAL ASTHMA AND HELMINTH INFECTIONS
Interestingly, after exposure to Asp, Retnla−/−
mice displayed a minor (but statistically significant) decrease in lung eosinophilia. Similar to Relm-α, arginase I is another hallmark gene of alternatively activated macrophages and is induced after allergen challenge and helminth infection. Although the cationic amino-acid transporter–2 and arginase I were shown to play key roles in response to helminth infection (45
), arginase I is not required for allergen-induced inflammation, AHR, or collagen deposition (47
). Furthermore (and similar to our data demonstrating a role for Relm-α only in IL-13–induced responses but not allergen challenge), RNA interference targeting arginase 1 abrogated the development of IL-13–induced AHR (48
), but not allergen-induced AHR (47
Collectively, these data suggest that the classic and major products of alternatively activated macrophages (Relm-α and arginase I) play key roles in helminth-induced immune responses, but limited roles in allergen-induced airway allergic inflammation (). This effect could be attributable to the lack of chronicity of allergen or antigen exposure. Although allergen exposure is rather limited, the exposure to helminth antigens driving the Th2 response may be more chronic. Because our experimental regimes were mainly conducted in models that mimic acute allergic airway inflammation, we cannot exclude the possibility that Relm-α may play a more important role after chronic exposure to allergens. In addition, the role of Relm-α may be dependent on its cellular source and its synergy with other secreted molecules that may be present in the inflammatory milieu, which may differ in allergic and helminth infection. For example, we recently established a proinflammatory and synergistic role for Relm-α in LPS-induced macrophage activation (27
). In the presence of LPS, Relm-α induced macrophage IL-6 and TNF-α secretion, while decreasing IL-10 secretion (27
). Thus, the role of Relm-α may be dependent on its synergism with LPS or other pattern recognition–dependent pathways, which may largely vary between allergic settings and parasitic infections. For example, recent studies highlighted key roles for low doses of LPS and Toll-like receptor 4 in allergenicity. In contrast to allergens, which require narrow (but necessary) innate immune activation (49
), helminth infections may activate numerous innate immune pathways, including various pattern-recognition receptors (e.g., Toll-like receptors, C type lectin receptors, protease-activated receptors, and nod-like receptors) (51
). In fact, parasites can activate multiple innate immune components via chitin, proteases, lectins, and secretory components (e.g., lacto-N
-fucopentaose III, schistosome-derived lysophosphatidylserine, and phosphorylcholine-rich glycoprotein), whereas the repertoire of innate immune activation by allergens is more limited (49
). It would be interesting to determine whether any of the aforementioned components up-regulate Relm-α expression and whether Relm-α regulates LPS-induced effects in the lungs. Moreover, given the proposed role of Relm-α in innate immune responses and in helminth-induced Th2 responses (23
), to assess its function in infection-associated asthma would be intriguing. For example, rats infected with localized pulmonary cryptococcal infection display increased IL-13 expression and consequent disease pathology (including AHR and mucus production) (60
). Thus, Relm-α may play a more significant role in disease settings that involve increased IL-13 responsiveness.
In conclusion, we demonstrate that Relm-α is secreted into the airway lumen and is differentially dependent on IL-13Rα1 and Type I IL-4 receptors after OVA and Asp challenges, respectively. The differing cellular sources of Relm-α after exposure to these two allergens are likely explained by the differential IL-4R requirement and the relative induction of IL-4 and IL-13 in response to distinct allergens (14
). Indeed, increased IL-13 concentrations will promote a predominant Type II IL-4R–dependent response pathway because IL-13–induced Relm-α expression mainly occurs in epithelial cells. Functionally, using Relm-α gene–targeted mice, we showed that Relm-α was largely redundant in terms of inducing Th2 cytokines, mucus, and inflammatory cell infiltration into the lung. These results mirror the dispensable role that other alternatively activated macrophage products (such as arginase 1) play in acute models of allergen-induced experimental asthma and contrast with their role in the setting of parasitic infections. These data suggest a divergence between allergen-induced responses and helminth-induced Th2-type immunity, based on our collective data concerning Relm-α.