Gut Microbiota Shape Lung Immunity See article p.97

Ron Regulates Reparative Macrophages See article p.256

Clarifying Complement with Crystals See article p.337

Nod2 Needed To Curb Intestinal Inflammation See article p.345

The humanized mAb eculizumab prevents the cleavage of complement component C5 into C5a and C5b and thus blocks both C5a-mediated inflammatory responses and initiation of the terminal complement pathway via membrane attack complex formation. Eculizumab has been approved to treat patients with the complement dysregulation diseases paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome, and a better understanding of its mechanism of action could be applied to augment its efficacy in these and other diseases. In this issue, Schatz-Jakobsen et al. (p. 337) solved the crystal structure (to 4.2 Å resolution) of C5 bound to an Fab fragment with the same sequence as eculizumab. Although the resolution of the structure did not allow determination of fine details, the variable domains of the Fab, including all but one CDR region, could be unambiguously shown to interact with the antiparallel four-stranded β-sheet of the MG7 domain of C5. This binding pattern led the authors to conclude that eculizumab sterically hinders the interaction of C5 convertases with C5 but does not directly block the scissile bond that is cleaved to form C5a and C5b. The residues of C5 bound by the Fab explained the high degree of specificity of eculizumab for human C5 and provided a basis for the observed resistance to eculizumab of a subset of PNH patients bearing polymorphisms in C5. To validate the conclusions drawn from the crystal structure, the authors introduced 66 single point mutations in the CDR residues of the Fab and then measured the effects of these mutations on binding kinetics to C5 and functional activity. Results were in agreement with predictions made from the crystal structure; in particular, mutating residues predicted to be important for binding reduced the ability of the Fab to inhibit complement activation. These data provide important insight into eculizumab mechanisms of action and have implications for the rapidly advancing field of complement-targeted therapeutics.

Chronic infection with Aspergillus fumigatus can cause severe pulmonary allergic disease, and T cell responses to this infection contribute to pathology that causes lung injury. In this issue, McAleer et al. (p. 97) examine how commensal bacteria in the gut can influence T cell responses in the lung during A. fumigatus infection. Mice colonized with segmented filamentous bacteria (SFB) in the gut were treated (or not) with water containing the antibiotic vancomycin for several weeks and then infected with A. fumigatus by oropharyngeal aspiration. The lungs of antibiotic-treated, A. fumigatus-infected mice had significantly lower levels of IL-17 and IL-22 and higher levels of IL-4 relative to those of infected mice not treated with vancomycin. IFN-γ levels and fungal lung burdens did not differ significantly between the two groups, suggesting that SFB in the gut influenced Th17 and Th2 responses. Vancomycin treatment was associated with decreased expression of genes encoding the antimicrobial proteins RegIIIβ and RegIIIγ in small intestinal but not lung tissue. RegIIIγ−/− mice showed greater Th17 priming in the lung upon A. fumigatus infection relative to wild-type (WT) infected mice, and this Th17 priming was associated with SFB colonization. IL-22 regulates expression of RegIIIβ and RegIIIγ genes in the intestinal epithelium, and IL-22−/− mice had significantly more weight loss during A. fumigatus infection than did WT mice. Intestinal treatment of IL-22−/− mice with recombinant RegIIIγ protein protected mice from weight loss during infection and reduced inflammatory cytokine production but did not directly impact SFB colonization, fungal burden, or IL-17 levels in the lung. In addition, i.v. serum transfer experiments indicated that IL-1 receptor ligands induced by commensal bacteria promote lung Th17 cell accumulation. Together, these data indicate that SFB colonization in the gut can influence pulmonary adaptive immunity.

Nod2, a cytosolic sensor of peptidoglycan, is expressed by numerous cell types and is critical to regulating host-microbiome interactions in intestinal tissue. Nod2 loss-of-function mutations have been identified in individuals with Crohn’s disease (CD), a condition in which chronic intestinal inflammation is associated with T cell activation. Zanello et al. (p. 345) now examine how Nod2 regulates mucosal damage in the small intestine. Wild-type (WT) and Nod2−/− mice showed significant mucosal damage in the small intestine immediately after treatment with anti-CD3 mAb to induce acute T cell activation. Mucosal damage persisted in Nod2−/− but not WT mice three days after treatment and included increased cell infiltration in the lamina propria, villous blunting, and shortening of small intestinal crypts. Relative to WT mice, Nod2−/− mice had a higher number of apoptotic cells in the mucosa, delayed epithelial cell regeneration in the small intestine, and a greater accumulation of IL-17A-expressing T cells in the lamina propria. Microbial load influenced the degree of intestinal tissue damage caused by anti-CD3 treatment in Nod2−/− mice, suggesting that microbial sensing is involved in tissue damage mechanisms. Mice engineered with specific deletions of Nod2 in intestinal epithelial cells or in Lyz2-expressing phagocytes did not show significant crypt damage relative to WT mice upon T cell activation. However, Nod2 deletion in Lyz2-expressing phagocytes was associated with more apoptotic epithelial cells and higher TNF-α and IL-22 expression. Together, these data indicate that Nod2 expression in phagocytic cells may contribute to controlling immune responses triggered by T cell activation that result in intestinal damage.

Chronic inflammation is associated with obesity and is mediated at least in part by the accumulation of inflammatory macrophages (M1) in tissues, accompanied by an imbalance of the ratio of M1 to alternatively activated, or reparative (M2) macrophages. The Ron receptor tyrosine kinase has been shown to inhibit the expression of genes encoding inflammatory mediators and may promote the differentiation of M2 macrophages, leading Yu et al. (p. 256) to assess whether Ron could play a protective role in obesity-related chronic inflammation. Relative to wild-type (WT) mice, Ron-deficient (Ron−/−) mice on a high-fat diet gained more weight and exhibited increased adiposity, increased fasting blood glucose, impaired glucose tolerance, and an increased ratio of LDL:HDL cholesterol. In WT mice, Ron expression was found in the relatively anti-inflammatory CD11c- resident adipose tissue macrophages (ATMs) much more often than in inflammatory CD11c+ ATMs, and expression in CD11c- ATMs decreased with weight gain. Supporting these indications of an anti-inflammatory, anti-obesity role for Ron, this kinase was found to have a protective role in models of atherosclerosis and hepatic steatosis. Relative to their ApoE−/− counterparts, aortas of Ron−/− X ApoE−/− double knockout (DKO) mice given a high-cholesterol diet (HCD) demonstrated increased lipid deposition and increased expression of genes encoding iNOS and numerous inflammatory cytokines. Analysis of total aortic macrophages in ApoE−/− mice revealed a decreased percentage of cells expressing Ron following exposure to a HCD and decreased expression of Arg1 in macrophages lacking Ron. Livers of DKO mice on an HCD had increased damage, lipid deposition, and expression of IL-12β and TNFα, as well as decreased Arg1 expression, compared with livers from similarly treated ApoE−/− mice. In ApoE−/− mice on a HCD, Kupffer cells expressing Ron upregulated Arg1, whereas those lacking Ron upregulated iNOS. Together, these data suggest that Ron is expressed on reparative macrophages and has a protective role in obesity and related inflammatory diseases. Further work will determine whether this kinase could be a useful target in promoting protection against the inflammatory consequences of obesity.