The protozoan Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis, which can be fatal for immunosuppressed or immunocompromised individuals. T. gondii expresses a profilin protein recognized by the endolysosomal receptors TLR11 and TLR12. This recognition initiates a cascade of events leading to dendritic cell (DC) production of IL-12, a response necessary for host defense. Previous work established that TLR11 recognized T. gondii profilin and was necessary for induction of protective IL-12. However, whether TLR11 does this alone or in concert with an accessory protein has not been established. Raetz et al. (p. 4818) clarified this question by demonstrating that TLR12 binds to T. gondii profilin directly and forms a heterodimer with TLR11. Both TLR11 and TLR12 colocalize with UNC93B1, a protein known to interact with TLRs and facilitate their transport and signaling. Profilin recognition by TLR11 and TLR12 induced DC IL-12 production through a mechanism requiring IFN regulatory factor 8 (IRF8) but not NF-κB. IRF8-expressing CD8+ DCs were determined to be the source of the protective IL-12. The authors concluded that host defense against T. gondii is achieved by parasitic profilin triggering TLR11 and TLR12 recognition, which leads to activation of the signaling molecule IRF8.
TNF-α Fazes Circadian Gene Timing
Complex interplay between the immune system and the circadian rhythm–controlling center of the brain, the suprachiasmatic nuclei (SCN) of the hypothalamus, makes teasing out mechanisms of how these two systems interact difficult. Astrocytes, brain cells that play a pivotal role in many functions of the CNS, participate in innate immune responses by secreting proinflammatory cytokines IL-1β, TNF-α, and IL-6, but whether these cells can contribute to regulation of SCN circadian rhythms by responding to immune stimuli is unknown. Duhart et al. (p. 4656) used TNF-α stimulation of SCN astrocytes from mice that express period 2 (Per2), an oscillating circadian “clock” gene that has a cyclical expression period of roughly 24 h, linked to a luciferase reporter (Per2luc) to determine whether the circadian response to immune stimuli is mediated at the SCN level. They found that TNF-α treatment induced primary SCN astrocytes to release CCL2, TNF-α, and IL-6 in culture and caused phase delays in Per2luc expression compared with astrocytes treated with vehicle alone. Administering conditioned media (CM) from SCN astrocyte cultures to NIH 3T3 cells caused increased promoter activity in Per1, a period gene related to Per2. Blocking TNF-α activity in CM with soluble TNF-αR prevented these changes. When delivered intracerebroventricularly in vivo, CM from primary astrocyte cultures also induced phase shifts in behavioral circadian rhythms and SCN activation in control mice but not TNF-αR mutant mice. These studies suggest that TNF-α can modulate circadian timing in astrocytes, which can respond to this inflammatory stimulus in turn by releasing cytokines that alter the cyclical expression of other circadian clock genes.
TLR4 Takes on Toxins
Hemolytic uremic syndrome (HUS) results from intestinal infection by Escherichia coli that produce Shiga-toxins (Stx). These bacteria cause outbreaks, particularly dangerous for children, in both developed and undeveloped areas. The endothelial damage in kidney and brain tissue results from Stx interacting with the globotriaosylceramide (Gb3Cer) receptor. Neutrophils have been shown to interact with Stx during HUS and have been proposed to sequester toxins and thereby protect endothelia while also causing further damage by transporting toxin from the intestine to the kidney. However, human neutrophils do not express the Gb3Cer receptor, so how they interact with Stx has remained a mystery. Brigotti et al. (p. 4748) solved this dilemma by identifying TLR4 as the human neutrophil receptor that recognizes both Stx1 and Stx2. They achieved this through competition and function experiments using LPS as an agonist and anti-TLR4 Abs as antagonists. Neutrophils challenged with LPS, Stx1, or Stx2 expressed the same pattern of cytokines at both the mRNA and protein levels. Thus, the authors have identified TLR4 as the receptor responsible for Stx-mediated activation of human neutrophils and provided a new therapeutic target for developing HUS treatments.
Minding the GAP in Asthma
Abr, a GTPase activating protein (GAP) that deactivates the Rho-family GTPase Ras-related C3 botulinum toxin substrate (Rac), regulates such processes as actin reorganization and NADPH oxidase expression in immune cells. Mice lacking Abr appear to be phenotypically normal, but exhibit exacerbated pathology in sepsis and pulmonary hypertension disease models. To determine whether Abr also plays a role in the development of allergic asthma, Gong et al. (p. 4514) employed the cockroach allergen (CRA) asthma model and found that immunizing and then challenging abr−/− but not wildtype (wt) mice i.n. with high doses of CRA proved fatal. Challenging mice i.n. with low CRA doses led to significantly increased airway hyperresponsiveness (AHR), mucus production, serum IgE, and leukocyte infiltration into the airways in abr−/− mice compared with wt mice. CD4+ T cell numbers and Th2 cytokines, IL-4 and IL-5, were also elevated in the lungs of abr−/− mice. Transferring CD4+ T cells from CRA-challenged abr−/−, but not wt, mice to naive wt recipients caused an increase in airway resistance when these mice received i.n. CRA, indicating a causal role for abr−/− T cells in this model. The authors found that these T cells had both increased levels of activated Rac-GTP and enhanced capability to chemotax toward CCL21, suggesting that Abr may restrict cell mobility by preventing Rac-dependent actin reorganization. These results demonstrate that Abr deficiency may contribute to increased AHR by dysregulating mechanisms that prevent Rac-dependent molecular events, allowing enhanced infiltration of CD4+ T cells into the airways during allergen challenge.
Tumor Regression Sans Side Effects?
The complex family of ErbB receptors regulates cell proliferation and differentiation and is commonly dysregulated during tumor formation. Deletion of any of the four evolutionarily conserved receptors is lethal to mouse embryos, indicating the importance of these receptors in tissue growth and regulation. T cell immunotherapy targeting ErbB has been effective against some cancers, including head and neck squamous cell carcinoma, ovarian cancer, and breast cancer. However, as with any immunotherapy, striking a balance between therapeutic efficacy against disease and toxic side effects is essential. Van der Stegen et al. (p. 4589) engineered chimeric receptor-bearing human T cells (T4+ T cells) that express both T1E28z, a chimeric Ag receptor (CAR) that redirects T cells to recognize relevant ErbB dimers, and 4αβ, a cytokine receptor that causes selective expansion of T cells following IL-4 stimulation. T4+ T cells effectively recognized mouse ErbB and were used in a SCID-Beige mouse model to evaluate preclinical toxicity. SCID-Beige mice were injected with human ovarian carcinoma or head and neck carcinoma cell lines that expressed luciferase. Both i.v. and intratumoral administration of T4+ T cells caused partial tumor regression with no detected clinical or histopathological toxicity. However, i.p. administration, while causing tumor reduction, also initiated the release of a potentially lethal, T4+ T cell dose–dependent “cytokine storm,” caused by T4+ T cells recognizing targets in both healthy and tumor tissues. IL-2 and IFN-γ were generated by human cells, while IL-6 was generated by mouse cells. Depletion of recipient macrophages prior to administration of T4+ T cells reduced the levels of both toxicity and the amount of secreted IL-6, implicating IL-6 as the effector in pathogenesis, which included weight loss, reduced mobility, piloerection, and death. Thus, the authors developed a unique model to test their ErB-targeted therapeutic product and determined that toxicity could be controlled through both the route of cell administration and the dose of T4+ T cells used in therapy.