Mutations that impair NADPH oxidase function have been linked to CGD (2
), and recent genetic studies have identified associations between ileal CD and genomic regions containing NADPH oxidase genes (9
). We investigated the role of the p40phox
subunit in intestinal homeostasis using gene-targeted mice and demonstrated that mice lacking the p40phox
subunit of NADPH oxidase are more susceptible to DSS-induced colitis. In addition, p40phox
-deficient mice showed enhanced neutrophil infiltration, a hallmark of both inflammatory bowel disease (IBD) and CGD (34
). We report the first evidence that NAPDH oxidase deficiency is crucial for the resolution phase, rather than the acute phase, of inflammation. Previous studies of DSS colitis that used genetically deficient mice for other NADPH oxidase subunits have reported different outcomes in their models (15
). Krieglstein et al.
demonstrated that the absence of p47phox
had no effect on colitis severity and colonic myeloperoxidase production after 7 days of 3% DSS treatment. Furthermore, through the use of an iNOS inhibitor, the investigators showed that in the absence of p47phox
and iNOS, animals were protected from DSS colitis (16
). On the other hand, Bao et al.
reported less colonic tissue damage and reduced myeloperoxidase levels after 7 days of 2.5% DSS in gp91phox
-deficient mice compared to WT (15
). Although these studies have reported that susceptibility to DSS colitis is not affected by p47phox
deficiency and is lessened in the absence of gp91phox
, we hasten to note that the recovery phase of inflammation was not investigated in these studies (15
In this study, we show that the absence of p40phox
mice enhances susceptibility to anti-CD40-induced colitis, suggesting that ROS in the innate compartment is essential during intestinal inflammation. However, in p40phox−/−
DKO mice, DSS-induced inflammation was less severe than in p40phox−/−
mice, implying a contribution from lymphocytes. Given that NAPDH oxidase activity has recently been shown to play a role in T cell differentiation and activation, p40phox
-deficient mice may have altered T cell responses in addition to the neutrophil phenotype. p47phox
deficiency has been associated with impaired T regulatory cell induction (44
) and increased IL-23 and TH
17 response (44
). Indeed, we observed 50% fewer peripheral T regulatory cells in p40phox−/−
mice than in WT mice (data not shown). While our data highlight an important role for p40phox
in neutrophils, we recognize that NAPDH oxidase activity may affect other components of the inflammatory response.
We demonstrate that neutrophil infiltration in p40phox−/−
mice is coincident with greater inflammation during DSS challenge. However, despite greater numbers of recruited lamina propria neutrophils in p40phox−/−
mice during DSS, the ROS-deficient phagocytes are unable to control infiltrating bacteria, as demonstrated by greater bacterial translocation to peripheral lymph nodes. This inability to efficiently kill microbes during DSS colitis may contribute to the exacerbated inflammation described in this NADPH oxidase-deficient model. During inflammation, ROS produced by neutrophils are thought to not only assist in anti-bacterial clearance but also to damage surrounding tissue (33
). However, mice deficient in the neutrophil chemokine Cxcl1 fail to recruit neutrophils during DSS colitis and show more severe symptoms, highlighting a role for neutrophils in restoring mucosal barrier integrity (46
). In order to address whether neutrophils are necessary for recovery from DSS- and/or anti-CD40-induced colitis, as well as to establish whether p40phox−/−
neutrophils are pathogenic, we performed in vivo
neutrophil depletion experiments. Depletion of WT neutrophils increased disease severity and induced 100% mortality by day 10 during DSS colitis and by day 4 during anti-CD40 colitis, suggesting that neutrophils are important in the resolution of intestinal inflammation. Neutrophil depletion in p40phox−/−
mice treated with DSS, however, induced no significant differences in weight loss and inflammation compared to their p40phox−/−
neutrophil-replete counterparts, suggesting that an inflammatory threshold had already been met in the absence of ROS. Previous reports using the RP-3 monoclonal antibody suggested that neutrophil depletion in WT rats suppresses DSS colitis; however, this antibody targeted the Gr1 antigen which consists of both Ly-6G and Ly-6C molecules and therefore depleted not only neutrophils but also a subset of macrophages. Thus, we propose that the observed differences may be due to the depletion of several immune populations in previous studies and/or the kinetics of antibody administration (47
). Our experimental design, in which a Ly-6G neutrophil-specific depletion antibody was repeatedly injected throughout the DSS course, differs from one-time depletion studies in which new neutrophils may be generated and recruited to assist in the resolution of inflammation at later stages of DSS. Using another anti-Gr1 antibody (RB6-8C5), Qualls et al.
demonstrated protection from DSS at early time points, which was lost by day 7 of treatment (48
). Thus we extend these reports by describing a role for neutrophils during the resolution phase of intestinal inflammation.
To identify disease-relevant pathways mediated by p40phox, we developed an integrative approach by combining functional studies of genetically deficient mice and murine models of IBD with cell-specific data extracted from clinical microarray studies of CGD. Bioinformatic analysis of the XCGD human PMN dataset revealed a signature in NFκB signaling, suggesting that NAPDH oxidase deficiency induces hyperactivity of this pathway. Data obtained from our murine model confirmed that proinflammatory cytokine expression was indeed higher in p40phox−/− mice than in WT mice during DSS colitis, correlating with the degree of inflammation in the colon. Thus, our model mimics human disease and demonstrates a role for p40phox in the regulation of proinflammatory mediators.
We identified a subset of genes whose expression is affected both by impaired NADPH oxidase activity in XCGD neutrophils and by inflammation in mouse colon. Given our in vivo
data implicating aberrant neutrophil recruitment, the neutrophil cell-specific analysis was key to unraveling the underlying mechanisms of increased colitis severity in p40phox
-deficient mice. Validating the findings from our computational analysis, we confirmed that during early DSS colitis, induction of the neutrophil chemokine receptor Ccr1
was significantly higher in p40phox−/−
colon compared to WT. Analysis of isolated lamina propria cells confirmed that in the context of NADPH oxidase deficiency, Ccr1 expression on neutrophils is significantly higher, despite equal levels of Ccl3
chemoattractants present in the colon. As Ccr1 plays a key role in neutrophil infiltration (32
), its upregulation in p40phox−/−
neutrophils likely accounts for the observed enhanced neutrophil recruitment in p40phox−/−
colon during DSS. However, despite their greater recruitment, p40phox−/−
neutrophils are unable to resolve intestinal inflammation. By using a Ccr1 antagonist in WT mice, we were able to block neutrophil recruitment to the colon during DSS colitis. Interestingly, we observed greater disease susceptibility in this setting, in agreement with a key role for neutrophils during healing. These data, as predicted by bioinformatic analyses, show not only that Ccr1 is regulated by p40phox
but moreover that neutrophils are necessary during the restitution phase of DSS.
Our integrated bioinformatic analysis further identified NADPH oxidase activity as a novel regulator of the enzymes involved in the modification of glycan structures during intestinal inflammation. Six genes identified through our integrative genomics approach (St3gal6
, and HexA
) were significantly impaired in p40phox−/−
mice during acute and recovery phases of DSS colitis. Moreover, by assessing these genes in neutrophil-depleted WT mice, we were able to demonstrate that p40phox
-intact neutrophils are required for their expression during DSS inflammation. Sialyltransferases and fucosyltransferases such as St3gal4, St6gal1, Fut4, and Fut9 are key regulators of leukocyte trafficking through selectin ligand formation. For example, St3gal4 promotes neutrophil adhesion (49
), while St6gal1 deficiency has been reported to induce neutrophilic inflammation (51
). Thus, decreased expression of sialyltransferases and fucosyltransferases in the absence of p40phox
may contribute to aberrant neutrophil trafficking. Furthermore, alterations in mucosal thickness and mucin structure have been reported in IBD and other mucosal disorders due to aberrant glycosylation (41
). St3gal6, St3gal4, Fut4, and Fut9 have been implicated in intestinal mucin modification and their impaired expression in p40phox−/−
mice may compromise the protective mucosal barrier integrity, resulting in enhanced inflammation (37
). Moreover, FUT4 contributes to epithelial wound repair during airway inflammation (59
), suggesting that its decreased expression in p40phox
deficiency may contribute to impaired resolution of inflammation. Together, our observations indicate that p40phox
deficiency leads to alterations in the expression of enzymes modifying glycan structures, which may induce aberrant intestinal neutrophil recruitment and impaired wound healing.
By combining functional studies in genetically engineered murine models with computational analysis, we have demonstrated that p40phox deficiency induces an aberrant neutrophilic inflammation in response to intestinal injury, likely due to alterations in the mechanisms controlling leukocyte recruitment and wound healing.