Our work provides new insight into the cellular mechanisms that drive M2 polarization during innate allergic inflammation in response to chitin. Our study suggests that macrophages are not M2 polarized directly on exposure to chitin in the lung. Instead, we demonstrate that chitin stimulates CCL2 production from airway epithelial cells, and we establish a pivotal role for CCR2 in chitin-induced macrophage polarization and innate allergic inflammation in the airway. Isolated CD326+ cells from chitin-exposed lungs and LA-4 epithelial cells exposed to chitin in vitro produced elevated levels of CCL2. Compared to WT mice, CCR2KO mice exposed to chitin demonstrated reduced expression of M2 markers (ArgI, CCL17 and CCL22), eosinophil recruitment and eosinophil activation in the lung. Thus, we propose that the respiratory epithelium modulates M2 polarization of macrophages upon chitin exposure.
Airway epithelial cells are among the earliest cells exposed to inhaled substances and actively collaborate with immune cells to mount innate and adaptive responses. Because chitin is associated with many inhaled allergens, we hypothesize that chitin recognition by airway epithelial cells may promote an epithelial pro-allergy program to otherwise innocuous agents via secretion of CCL2. Multiple lines of evidence underscore the association of CCL2 with asthma and allergic airway disease. Elevated CCL2 levels are present in BALF obtained from individuals with asthma (34
) and CCL2 is elicited upon airway allergen challenge in humans (35
). In animal models, OVA/alum sensitization and challenge provokes increased CCL2 expression (36
). Similarly, chitin-associated cockroach (37
) or Aspergillus fumigatus
) also provoke CCL2 production in animal asthma models. Our findings suggest that epithelial cells may be an important source of CCL2 during exposure to chitin-containing allergens, thereby promoting M2 polarization.
Consistent with a role for airway epithelial cell CCL2 in allergic responses, chitin exposure promotes M2 polarization in vivo
via an indirect mechanism (10
) that is dependent on CCR2. We propose that airway epithelial cell-derived CCL2 is a key chemokine in promoting M2 polarization and innate allergic inflammation in response to chitin. Indeed, CCL2 expression precedes ArgI expression in chitin-exposed lungs. Following chitin exposure, antibody neutralization of CCL2 in chitin-exposed epithelial cell supernatants also inhibits M2 polarization in vitro
. Airway epithelial cells are an important source of CCL2 in viral infections associated with asthma exacerbations (39
). Our findings that CCL2 promotes M2 polarization in a setting of chitin-induced allergic inflammation are consistent with previous reports demonstrating a role for CCL2 in tumor- (41
) and thermal injury- (25
) associated M2 polarization.
We observed that epithelial cell CCL2 is necessary for M2 polarization in response to chitin in vitro
, however CCL2 was dispensable for M2 polarization in vivo
. The upregulation of the CCR2 ligand, CCL7, in CCL2-deficient mice following chitin exposure may obscure the necessary role of CCL2 in chitin-induced M2 polarization we observed in vitro
. To address this issue, we investigated the host response to chitin in a CCR2-deficient mouse. In the absence of CCR2, chitin exposure failed to elicit alternatively activated macrophages in the lung. Our results reflect the established central role of CCR2 in mediating the effects of both CCL2 and CCL7 in vivo
. For example, in a model of infection with the fungal pathogen Histoplasma capsulatum
, CCR2KO mice demonstrated increased susceptibility to the fungus and an increased fungal burden in the lung (42
). Neutralization of either CCL2 or CCL7 was not sufficient to increase the fungal burden, however neutralization of both chemokines did. Similarly, CCR2KO mice are more susceptible to Listeria monocytogenes
infection whereas CCL2KO or CCL7KO mice demonstrate an intermediate susceptibility phenotype (30
). Moreover, M2 polarization canonically depends on IL4Rα and its ligands IL4 and IL13. While neither CCL7 nor IL4 or IL13 were detected following epithelial cell exposure to chitin in vitro
, all three products are present in the lung following chitin exposure in vivo
. Our work does not preclude a role for IL-4 in chitin induced innate allergic inflammation. However, future studies should address whether the CCL2-CCR2 axis and IL-4Rα signalling represent linear, convergent, or parallel signaling pathways in chitin-initiated M2 polarization.
CCR2KO mice also demonstrate reduced eosinophil recruitment and activation in response to chitin, further suggesting a role for CCR2 in eosinophilic inflammation. Murine eosinophils contain CCR2 mRNA (43
) and may express low levels of CCR2 protein on their cell surface (44
). However, this is unlikely to explain the reduction in eosinophil recruitment in CCR2KO mice as eosinophils do not migrate in response to CCL2 (45
) and migrate toward CCL7 via a CCR3-dependent mechanism (46
). Chitin induced eosinophil recruitment is abrogated in mice lacking the receptor for LTB4
), suggesting a link between eosinophil recruitment and leukotriene signaling following chitin exposure. Because chitin fails to activate eosinophils directly (47
), the defect in activation and recruitment in CCR2KO mice must be linked to upstream factors. M2 macrophages elicited by chitin exposure are potent sources of LTB4
). Thus, the reduction in chitin induced eosinophil recruitment in CCR2KO mice may be due to the reduction in M2 polarization.
CCR2 is required for egress (48
) and homing (49
) of monocytes to sites of inflammation. We show a near-complete reduction of Ly6Chi
monocytes recruited to the lung following chitin exposure in CCR2KO mice. Ly6Chi
monocytes may differentiate into CD11b+ inflammatory DCs, which accumulate in the allergic airway and are critical mediators of Th2
adaptive immune response in the airway (50
). Recently, CCR2+Ly6Chi
monocytes were reported to recruit eosinophils to colonic mucosa via CCL11 in an experimental colitis model (51
). The partnering of Ly6Chi
monocytes with M2 macrophages to recruit and/or activate eosinophils could represent an intriguing interaction to therapeutically target in order to reduce eosinophilic inflammation during asthma and allergy.
In conclusion, we show that airway epithelial cells secrete CCL2 and that CCR2 signaling is required for events leading to M2 polarization and recruitment and activation of eosinophils upon chitin exposure. The findings clarify the mechanisms that drive alternative activation of macrophages in response to chitin, and identify possible targets of therapeutic intervention in the setting of chitin induced innate allergic inflammation.