NETs Full of Bacteria
NETosis is a form of cell death used by neutrophils to elim-inate pathogens via the generation of neutrophil extracellular traps (NETs). During NETosis, neutrophils release genomic DNA and cytotoxic proteins into the extracellular space, forming structures that entrap and kill pathogens. To investigate the involvement of surfactant protein D (SP-D) in neutrophil antibacterial activity, Douda et al. (p. 1856) employed two mouse models of airway NET formation. In acute lung inflammation, the airways of LPS-treated mice showed a remarkable recruitment of neutrophils associated with high concentrations of soluble DNA. The pulmonary NETs appeared as string-like structures containing neutrophil elastase and cathelicidin, and citrullinated histone H3, a marker of NETs. SP-D was found to bind simultaneously to carbohydrate ligands and NET DNA fragments. Similar results were obtained in a model of chronic lung infection with Pseudomonas aeruginosa, demonstrating that NETs are formed in vivo. Finally, the authors explored the biological significance of the SP-D–DNA interaction. Scanning electron microscopy analysis showed that SP-D caused microagglutination of the bacteria, allowing them to be more efficiently trapped by NETs than nonagglutinated bacteria. This study provides evidence for a potential function of SP-D as an antibacterial protein.
Sepsis, Cancer, and Mortality
Apoptosis of immune populations has been implicated as a contributing factor to septic lethality based on experiments performed in mouse models using previously healthy animals. Because sepsis is prevalent among critically ill patients, such as cancer patients, Fox et al. (p. 1950) investigated whether prevention of lymphocyte apoptosis would improve the survival of tumor-bearing mice in a pneumonia model of sepsis. Transgenic mice (Bcl-2-Ig) overexpressing bcl-2 in T and B lymphocytes were challenged with pancreatic adenocarcinoma and then infected with Pseudomonas aeruginosa, causing development of sepsis in the setting of pre-existing cancer. Interestingly, although the septic mice had decreased levels of lymphocyte apoptosis, their mortality rates were significantly higher compared with the wild-type mice. In addition, the mice had increased levels of pulmonary inflammatory cytokines, but pneumonia severity, lung inflammation, and tumor size were similar between transgenic and wild-type mice. The survival results were confirmed in another sepsis model of decreased apoptosis, as Bim−/− mice with pre-existing cancer had increased mortality after development of pneumonia. Thus, the authors cautioned that, in some settings, blocking lymphocyte apoptosis in septic patients with comorbidities might actually worsen patient prognosis.
Fibrin Flusters Infection
Activation of the coagulation system during infections has the potential to either contribute to deleterious coagulopathies or exert a protective effect. To develop safe anti-infection therapeutics, it is critical to gain an in-depth understanding of the regulation of the hemostatic process. Luo et al. (p. 1866) studied the role of several coagulation factors in the host response against sublethal concentrations of the bacterium Yersinia enterocolitica. Compared with control infected mice, i.p. or systemically infected fibrinogen-deficient mice and Coumadin-treated mice, which generate low levels of fibrin, had significantly increased bacterial burden and decreased survival. Mice expressing low levels of tissue factor (TF) were susceptible to the infection, with a mortality rate comparable to the fibrinogen-deficient mice. A similar phenotype was displayed by mice deficient in both plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor. In contrast, factor XI-deficient mice were resistant to infection. Because the extrinsic coagulation pathway is initiated by TF, whereas the intrinsic pathway is TF-independent and factor XI-dependent, the data support an important protective role for fibrin and the extrinsic coagulation pathway in host resistance to Y. enterocolitica.
The resolution phase of acute inflammation at the tissue level is a tightly controlled process involving the generation of lipid mediators from polyunsaturated fatty acids. For instance, docosahexaenoic acid (DHA) is the precursor of the D series of resolvins, which includes aspirin-triggered resolvin D1 (AT-RvD1), its precursor 17(R)-hydroxy DHA (17R-HDHA), and resolvin D2 (RvD2). In this issue, Bento et al. (p. 1957) report the protective effect of members of the D resolvin family in two mouse models of experimental colitis. Treatment of the animals with 17R-HDHA, AT-RvD1, or RvD2 resulted in significant reduction of disease activity index, body weight loss, and colon damage both in dextran sulfate sodium- and trinitrobenzene sulfonic acid-induced colitis. Amelioration of colonic inflammation in the resolvin-treated mice was associated with decreased infiltration of neutrophils into the colon, reduced levels of the proinflammatory mediators TNF-α, IL-1β, MIP-2, and CXCL1/KC, lower mRNA expression of adhesion molecules and COX-2, and inhibition of the NF-κB pathway. Lastly, the authors found that blocking the interaction of AT-RvD1 with its receptor ALX in vivo prevented the protective effect of the lipid mediator. Thus, resolvins represent attractive candidates for the development of therapeutics aimed at combating intestinal inflammation.
TNFR1 Blockade Beats Diabetes
TNFR1 deficiency protects NOD mice from type 1 diabetes (T1D). The aim of the study by Chee et al. (p. 1702) was to investigate the mechanism of this protection. The authors studied T1D incidence and insulitis in TNFR1-deficient NOD mice (NOD/TNFR1−/−) and in NOD/TNFR1−/− mice crossed to diabetes-susceptible TCR transgenic NOD4.1 and NOD8.3 mice, which have diabetogenic islet-specific CD4+ and CD8+ T cells, respectively. All of these mice were significantly protected from T1D and insulitis compared with wild-type controls, indicating that the absence of TNFR1 signaling affected both CD4+ and CD8+ T cells. Additional studies showed that TNFR1 signaling was not required for beta cell death or maturation of dendritic cells or proliferation of islet Ag-specific T cells. Interestingly, TNFR1 deficiency resulted in defective homing of T cells into pancreatic islets and increased the number of regulatory T cells (Tregs). Moreover, Tregs from TNFR1-deficient mice had suppressor function that was superior to that of wild-type Tregs. In conclusion, blockade of TNFR1 signaling appears to advantageously affect Treg function and inhibit the onset of diabetes, supporting the use of anti-TNF therapeutics in T1D.
Losing Sight with IL-17
T cells are pivotal players in the induction of herpes simplex virus (HSV)-induced ocular stromal keratitis (SK). Suryawanshi et al. (p. 1919) investigated the involvement of IL-17 in SK pathology using a mouse model of ocular HSV infection. Corneal IL-17 expression was upregulated in a biphasic manner, with an early release from γδ T cells and later production by Th17 cells. Kinetic analysis of the cells present in the infected corneas revealed that Th1 cell numbers peaked at day 15 postinfection and then declined. In contrast, the number of Th17 cells gradually increased with the disease progression. This increase was conceivably due to the establishment of a Th1-induced microenvironment that promoted the generation and/or migration of Th17 cells into the cornea. Indeed, at day 15, substantial concentrations of IL-6, TGF-β, and CCL20 were detected in the infected eyes, whereas production of IFN-γ and IL-2 dropped. The contribution of IL-17 to SK pathology was substantiated by treating the infected mice with an IL-17–neutralizing Ab. This treatment resulted in significantly diminished SK lesion scores, lower frequencies of Th1 and Th17 cells, and reduced neutrophil infiltration. A similarly reduced disease severity was observed in IL-17R KO mice. This study thus indicates IL-17 as a potential therapeutic target for the treatment of SK.
Effective anti-cancer mAbs engage activatory FcγRs on effector cells; consequently, cytotoxic mAbs are often modified to improve their interaction with cells expressing FcγRs. In mice, the IgG2a isotype has high binding affinity for activatory FcγRs and is used in tumor models. White et al. (p. 1754) investigated the isotype requirements for the function of immunostimulatory mAbs that exert their activity through interactions with immune receptors such as CD40. The authors engineered the variable regions of the rat anti-CD40 mAb 3/23 onto the murine IgG constant regions of different isotypes, generating the mAbs 3/23 m1 (IgG1) and 3/23 m2a (IgG2a), which bind to distinct FcγRs. Although both mAbs were biologically active, only 3/23 m1 stimulated B and T cell responses and induced strong activation of APCs. Using knockout mouse strains that selectively lack the FcγRIIB receptor (FcγRIIB−/−) or all activatory FcγRs (FcγR−/−), the authors ascertained that the immunostimulatory activity of the anti-CD40 mAb was mediated by binding to FcγRIIB, normally considered an inhibitory receptor. Additional studies demonstrated that the function of this receptor was to mediate mAb crosslinking. This study highlights the importance of understanding the mechanism of action of individual mAbs as a means to engineer more potent variants and improved therapeutics.
Phospho SQ Not Needed
V(D)J recombination in lymphocytes is initiated by the RAG endonuclease, a complex formed by RAG1 and RAG2 proteins, which binds to and cleaves the DNA. The cleaved DNA is repaired by enzymes through ligation of DNA coding ends and signal ends to form coding and signal joints, respectively. The PI-3–like kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs are activated in the recombination process and specifically phosphorylate protein motifs consisting of serine and threonine residues followed by glutamine residues (SQ/TQ). ATM and DNA-PKcs are known to regulate DNA repair, although their substrates have not been definitively identified. Gapud et al. (p. 1826) investigated whether RAG1 and RAG2 are the targets of these kinases, as they contain multiple SQ/TQ motifs and have been implicated in the repair process. The authors used an experimental system complementing RAG1- or RAG2-deficient Abelson transformed pre-B cell lines with retroviral constructs containing wild-type proteins or RAGs with mutated SQ/TQ motifs. On the basis of their results, the authors conclude that phosphorylation of RAG1 and RAG2 is not necessary for chromosomal coding or signal joint formation. The authors’ experimental approach is representative of in vivo V(D)J recombination and may be useful for further analysis of RAG mutations and activity.
Summaries written by Bernardetta Nardelli, Ph.D.