TLR activation triggers a variety of innate and adaptive immune responses, and some evidence suggests that TLR signaling may be linked to neurological responses. Qi et al. (p. 6417) explored this link by examining the effects of stimulating TLR3, TLR7, or TLR9 on pain responses in dorsal root ganglion neurons (DRGNs). Immunofluorescence staining showed that all three of these TLRs were expressed on both murine and human DRGNs, and stimulation of murine DRGNs with TLR3, TLR7, or TLR9 ligands specifically increased each receptor's expression. Relative to unstimulated DRGNs, TLR3, TLR7, or TLR9 stimulation increased expression of proinflammatory mediators that are associated with pain hypersensitivity, including IL-1α, IL-1β, and PGE2. The transient receptor potential vanilloid type 1 (TRPV1), a receptor known to respond to pain stimuli, also displayed increased expression and calcium flux in DRGNs treated with these TLR ligands. These observations were supported in a mouse model of pain in which treatment with a TLR9 antagonist reduced temperature sensitivity in mice implanted with tumor cells near the sciatic nerve. Overall, these findings suggest that stimulation with specific TLR ligands can be associated with nerve pain.

One factor that may contribute to differences in overall health observed in females versus males is cellular X chromosome mosaicism. Chandra et al. (p. 6465) examined the effect of mosaicism for X-linked gp91phox deficiency on sepsis in female mice. gp91phox, the catalytic subunit of the NADPH oxidase complex, is involved in generating superoxide anions that are used for antibacterial responses. The cecal ligation and puncture model of sepsis was used to compare female mice that were gp91phox-deficient, heterozygous mosaics or wild-type (WT) littermate controls. Sepsis-induced mortality was reduced in mosaic and deficient mice relative to WT mice, although bacterial levels in the blood were highest in deficient mice. Among these different mouse strains, sepsis induced a variety of changes in cytokine responses and in the white blood cell composition of the blood and bone marrow. Within the mosaic mice, gp91phox-deficient neutrophils had higher membrane expression of CD11b and greater organ infiltration than did gp91phox-expressing neutrophils. In vitro induction of an oxidative burst in neutrophils from mosaic mice increased oxidant levels in gp91phox-deficient neutrophils, presumably from oxidants released by WT neutrophils. These results suggest that gp91phox mosaics do not show a phenotype midway between those of deficient and WT mice, but instead have several characteristics that provide a protective advantage against sepsis.

An understanding of the effects of aging on T cell immunity is hindered by the logistical challenges of collecting data over time in human subjects, necessitating alternative approaches, such as computational modeling of these responses. Naumov et al. (p. 6617) studied the influenza-specific CD8+ T cell clonotypes generated in a middle-aged and an older human cohort and applied these observations to agent-based modeling of aging responses. The number of observed clonotypes in these cohorts was measured in PBMC cultures restimulated with the M158–66 influenza peptide. Clonotype responses were polyclonal in both cohorts, as identified by CDR3 nucleotide sequences, but older individuals had fewer clonotypes, especially those that occurred at lower frequency. With these clonotype data, an agent-based model was developed to predict aging repertoires in silico. A model of selected expansion with senescence, which accounted for the initial clonotype frequency, best fit the clonotype distribution observed in the older cohort. Overall, these results deepen our understanding of T cell aging and bring attention to the potential use of mathematical modeling of immune responses.

The inflammation associated with asthma is sustained in part through the accumulation of eosinophils and T cells in the airway, but little is known about how this inflammation is resolved. Haworth et al. (p. 6129) have defined a role for NK cells receptive to the lipid mediator resolvin E1 (RvE1) in the resolution of airway inflammation. A self-limited murine model of allergic airway inflammation revealed that eosinophils and T cells decreased in the lungs and mediastinal lymph nodes (MLNs) as inflammation resolved upon termination of allergen exposure. At the same time, NK cells accumulated in these sites, and NK cells in the MLNs expressed activation markers. Depletion of NK cells during the resolution phase correlated with persistent inflammation and decreased clearance of eosinophils and allergen-specific CD4+ T cells. CXCR3 and CD62L expression by NK cells was involved in homing to inflamed sites, and expression of NKG2D ligands on allergen-specific CD4+ T cells and eosinophils was required for NK cell-mediated clearance. NK cells also expressed the receptor for RvE1. NK cell depletion abrogated RvE1-mediated inflammation resolution, and in vitro treatment of NK cells with RvE1 enhanced their cytolytic function. These results provide a unique perspective of the roles that NK cells and RvE1 play during the resolution of airway inflammation.

The inflammation associated with inflammatory bowel diseases (IBD) like colitis results in the breakdown of the epithelial barrier lining the colon. Kominsky et al. (p. 6505) used metabolomic analyses and a murine model of dextran sulfate sodium (DSS)-induced colitis to better understand the effects of IBD on metabolic processes. Magnetic resonance spectroscopy analysis of murine colonic tissues from DSS-treated mice or a human intestinal epithelial cell line revealed induction of inflammatory responses correlated with changes in methylation-associated metabolites, relative to untreated controls. Further analysis confirmed that inflammation induced several key enzymes involved in methylation, including S-adenosylmethionine synthase and S-adenosylhomocysteine (SAH) hydrolase, and caused increased DNA methylation, compared with untreated cells. In comparison with DSS-treated mice, pretreatment with an SAH hydrolase inhibitor significantly inhibited NF-κB activity and caused more severe colitis symptoms. Conversely, augmentation of methylation by folate supplementation of DSS-treated mice reduced colitis symptoms relative to control mice. These observations reveal methylation and NF-κB activation as important mechanisms for protecting colonic epithelial cells against excessive inflammation and IBD.

Murine studies have revealed that platelet-activating factor (PAF) contributes to anaphylaxis by causing increased vascular permeability and vasodilation. PAF is thought to alter vascular permeability by inducing NO production via endothelial nitric oxide synthase (eNOS) through generation of phosphatidylinositol 3,4,5-triphosphate (PIP3) upon activation of PI3K/Akt signaling. The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) negatively regulates PI3K signaling through dephosphorylation of PIP3. Kang et al. (p. 6625) have examined how PTEN influences PAF-induced anaphylaxis. Relative to untreated mice, PTEN activity decreased rapidly in the lungs of mice upon anaphylaxis induced by either ovalbumin challenge or injection of PAF, and this was accompanied by increased PI3K activity and eNOS and Akt phosphorylation. Anaphylaxis symptoms were reduced and PI3K/Akt signaling responses decreased in mice pretreated with a recombinant adenovirus expressing PTEN. Decreased PTEN activity during anaphylaxis was linked to PTEN phosphorylation via casein kinase 2 (CK2), and PAF treatment increased CK2 activity. PAF-induced PTEN phosphorylation was inhibited and anaphylaxis was attenuated in mice treated with a CK2 inhibitor. These results provide a mechanistic insight into the signaling events surrounding PAF-mediated anaphylaxis and suggest new targets for therapeutic intervention.

TCR stimulation triggers signaling events essential to T cell activation, including activation of functional LFA-1 integrin molecules that help form T cell:APC conjugates. Integrin activation has been shown to involve the adhesion and degranulation promoting adapter protein (ADAP), which can associate with Src kinase-associated phosphoprotein (SKAP55), and ADAP has also been shown to promote NF-κB activation. Burbach et al. (p. 6227) examined how the association of SKAP55 with ADAP alters ADAP function using a chimeric ADAP/SKAP55 molecule. T cell:APC conjugates formed by ADAP−/− T cells expressing the ADAP/SKAP55 chimeric molecule were comparable to the level of conjugates formed by wild-type T cells. Mutagenesis analysis identified the amino acid arginine 131 in the pleckstrin homology domain of SKAP55 as required for interactions with and activation of LFA-1 integrin complexes. NF-κB signaling was not restored in ADAP−/− T cells expressing the SKAP55/ADAP chimera, despite the presence of sequences within ADAP known to be required for NF-κB activation. In contrast, expression of a SKAP55/ADAP chimera with methionine in place of arginine at position 131 rescued NF-κB signaling in ADAP−/− T cells. These data suggest that LFA-1 activation by ADAP is driven by complex formation mediated by the SKAP55 pleckstrin homology domain, and this interaction reduces the availability of ADAP molecules able to activate NF-κB signaling.

Apoptosis and caspase-1–mediated pyroptosis are cell death processes in which the hemichannel pannexin-1 is thought to play a role. Qu et al. (p. 6553) generated pannexin-1–deficient mice (Panx1−/−) to better understand the function of this molecule during cell death. ATP stimulation of the purinergic receptor P2X7R has been suggested to induce pannexin-1 pore formation, which allows entry of bacterial products into the cytoplasm and inflammasome-mediated activation of caspase-1. This mechanism was tested in vitro in bone marrow-derived macrophages from wild-type (WT) and Panx1−/− mice primed with LPS and treated with stimuli known to activate different types of inflammasome complexes. Pannexin-1 was not required for caspase-1 activation by any of these inflammasomes or caspase-1–induced secretion of IL-1β and IL-18. Pannexin-1 was also dispensable for ATP-induced P2X7R pore formation. In contrast, Panx1−/−thymocytes undergoing apoptosis were less able to take up YO-PRO-1, a nucleic acid dye, compared with WT or P2rx7−/−- thymocytes, and released less ATP relative to WT thymocytes. Apoptotic Panx1−/− thymocytes were also unable to promote migration of macrophages in a transwell assay. These results indicate that pannexin-1 is involved in ATP release and the production of “find me” signals that are essential to recognition and phagocytosis of apoptotic cells.

Summaries written by Christiana N. Fogg, Ph.D.