C-rac-king the Code of Smoke-induced Pneumonia Susceptibility

Pneumonia causes a tremendous burden of disease, impacting more individuals than HIV, cancer, diabetes, and many other leading public health priorities (1). Although impressive, these burdens are underestimates, because respiratory infections and their resulting host countermeasures influence the occurrence and/or severity of virtually all lung diseases including cancer and chronic obstructive pulmonary disease, conditions with well-established ties to tobacco smoking (1, 2). The threat of pneumonia is also much greater among smokers (3, 4). As many as one in three pneumonia cases has been attributed to smoking (3), which represents pneumonia’s most significant modifiable risk factor (4). Given the importance of lung infections and the degree to which they coalesce with other diseases (5), discovering mechanistic connections between cigarette smoke and pneumonia has important and broad clinical implications.

Pneumonia outcome is dictated by integrated signals controlling immune resistance and tissue resilience, with the former relying on contributions from numerous cellular sources such as lung epithelium and the resident and recruited leukocyte pools (5). Many of these cell types can be adversely affected by cigarette smoke (2), but precisely how this drives pneumonia susceptibility remains unclear. In this issue of the Journal, Larson-Casey and colleagues (pp. 1288–1301) reveal a compelling connection between macrophage NOX2 (NADPH oxidase) activity and heightened pneumonia vulnerability in the setting of cigarette smoke (6). Knowing that macrophages are essential for pulmonary host defense (7), due in part to NOX2-derived reactive oxygen species (ROS) (8), the authors investigated the effects of smoking on macrophage responses and host outcome in a mouse model of pneumococcal pneumonia. Cigarette smoke exposure markedly impaired clearance of Streptococcus pneumoniae from the lungs, and this finding was associated with a complete loss of infection-induced ROS in BAL cell membrane fractions. Importantly, pneumonia-induced cytokines and BAL leukocyte accumulation were unaffected by cigarette smoke, suggesting that immunodeficiency was likely a consequence of diminished cellular function rather than limited recruitment.

While investigating potential effects of cigarette smoke on the NOX2 complex, the authors found that p67^phox and Rac2 (Rac family small GTPase 2), which is required for the former’s membrane translocation, were both reduced after smoke exposure. Substantial in vitro and in vivo evidence supports their conclusion that abrogated Rac2 responses are causally linked to impaired defense in this setting. Pneumococcal infections in Rac2^−/− mice phenocopied the effects of cigarette smoke to a remarkable degree, eliminating BAL cell membrane ROS generation, and reducing bacterial killing and survival with no appreciable changes in leukocyte recruitment or inflammatory cytokine induction. This finding alone does not firmly establish Rac2 as the functional liaison between smoke and immunodeficiency. However, the fact that cigarette smoke exposure failed to elicit measurable effect in Rac2^−/− mice lends strong support to this hypothesis, as does their finding that Rac2 overexpression in mouse lungs (predominantly macrophages) completely reversed immunodeficiency due to either smoke exposure or Rac2 deficiency. To begin addressing mechanisms whereby cigarette smoke promotes the loss of Rac2, the authors measured a panel of smoke-associated metals in mouse BAL fluid and identified cadmium as the sole elevated representative. Impressively, cadmium administration, like Rac2 deficiency, recapitulated the effects of smoke during pneumonia, and mechanistically, the authors’ data suggest that both cigarette smoke and cadmium target Rac2 by limiting isoprenylation at C-189, a requisite post-translational modification for Rac2 function.

Overall, the use of multiple complementary approaches elegantly supports mechanistic relationships among smoking, cadmium, Rac2 deficiency, and pneumonia susceptibility, but whether this finding is applicable to human disease is a critical albeit more difficult question to address. To this end, the authors found that cadmium concentrations were significantly and selectively (vs. other metals) elevated in BAL cells from human smokers, and this finding again correlated with substantial loss of membrane Rac2 and p67^phox localization and ROS synthesis. The proposed importance of Rac2-mediated immunity is consistent with rare observations in children bearing dominant negative Rac2 mutations, which is associated with immunodeficiency similar to that observed in patients with leukocyte adhesion deficiency and chronic granulomatous disease (9). The current investigations raise exciting possibilities for new research directions aimed at unveiling the functional relationship between Rac2 and pneumonia in smokers.

Another remaining question pertains to the macrophage-specific nature of the authors’ findings. Their data implicate macrophages and not neutrophils as the predominant site of Rac2 dysfunction after smoke exposure, because depletion of the former but not the latter diminished ROS formation and antibacterial defense. This is surprising given the long-established role of neutrophils in lung immunity, both clinically and experimentally (10, 11). Indeed, Rac2 itself has been linked to neutrophil-derived IFN-γ and antibacterial defense in the lungs of mice challenged with pneumococcal pneumonia (12), whereas the experimental circumstances of the present study do not indicate an essential role for this cell type. It will be interesting and important to determine whether these findings in macrophages extend to other cell types, including but not limited to neutrophils, as experimental conditions vary. Even among macrophages themselves, both resident and recruited, functional heterogeneity (13) demands a more comprehensive interrogation of macrophage subsets. Last, and notably, others have reported increased oxidative stress in macrophages exposed to cigarette smoke (14, 15), possibly as a source of lung tissue damage. Although this seemingly contradicts the results of the current study by Larson-Casey and colleagues, differences could certainly be attributable to where (membrane vs. total) and/or how such observations were made, with responses to live bacteria after smoke exposure being entirely different from those recorded under alternative conditions.

This work represents a significant advance for a staggeringly harmful relationship that has long frustrated lung researchers and clinicians. The mechanistic basis of pneumonia susceptibility in smokers is surely more complex than the effect of one metal (cadmium) on one biological process (NOX2 activity) in one cell type (macrophages). Yet, the authors have provided a compelling piece of the puzzle and an interesting platform for future investigations.