Nucleotide-binding, oligomerization domain (NOD)-like receptors (NLRs) are a family of cytosolic proteins that play an important role in inflammation and immunity. The NLR family consists of twenty-two human proteins and at least thirty-four mouse proteins that contain a central nucleotide-binding, oligomerization domain (NACHT/NOD), an N-terminal protein interaction domain, and a variable number of C-terminal leucine-rich repeats.1 Similar to the toll-like receptors (TLRs), many well-characterized NLRs have been shown to function as pattern recognition receptors. These NLRs detect specific pathogen-associated molecules or endogenous damage signals and initiate the innate immune response. NOD1 and NOD2 detect components of bacterial peptidoglycan and recruit signaling molecules that drive the NF-kappa B-/AP-1-dependent expression of pro-inflammatory cytokines. Other NLRs, including NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4/IPAF, and NAIP, are activated by a variety of agonists and have been reported to form large, multiprotein inflammasome complexes that serve as platforms for the cleavage and activation of Caspase-1.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 Caspase-1 activation subsequently leads to the processing and secretion of IL-1 beta and IL-18 and may induce an inflammatory form of cell death known as pyroptosis.
In contrast to NOD1, NOD2, and some of the inflammasome-associated NLRs, many members of the NLR family have not been widely investigated. Of these, a small group consisting of NLRC3, NLRP2, NLRP4, NLRP7, NLRP12, NLRX1, and NLRP10/PYNOD has been suggested to have anti-inflammatory activities due to their ability to either negatively regulate NF-kappa B signaling or inhibit Caspase-1-mediated IL-1 beta secretion.17, 18, 19, 20, 21, 22, 23 As the only NLR that lacks the leucine-rich repeat domain at its C-terminal end, NLRP10 is of particular interest because initial studies using overexpressed NLRP10 or Nlrp10 transgenic mice indicated that it may negatively regulate inflammasome activation.18, 24
Several recent reports now challenge the hypothesis that Nlrp10 functions as an anti-inflammatory NLR and suggest that it may have a novel, inflammasome-independent function that is required for the adaptive immune response.25, 26, 27 The first report of this unique activity came from a study by Eisenbarth et al., who investigated the immune response to multiple agonists in Nlrp10-deficient mice.25 Since previous studies showed that Nlrp10 regulates cytokine secretion induced by inflammasome activation or NF-kappa B signaling, the authors tested this hypothesis using Nlrp10-/- macrophages. In contrast to Nlrp3-/- macrophages, wild-type macrophages and macrophages from Nlrp10-/- mice secreted comparable levels of IL-1 beta in response to Nlrp3 agonists. Additionally, Nlrp10-/- macrophages secreted similar levels of IL-6 and TNF-alpha as both Nlrp3-/- macrophages and wild-type macrophages following stimulation with different TLR agonists. Together, these observations suggested that Nlrp10 is not required to regulate Nlrp3 inflammasome activity or the NF-kappa B-dependent expression of other innate pro-inflammatory cytokines. Although cytokine levels were not affected by the absence of Nlrp10, Nlrp10-/- mice displayed a significant defect in their ability to activate the adaptive immune response.25 This defect was revealed following sensitization and challenge of Nlrp10-/- mice with multiple adjuvants aimed at inducing a T helper type 1 (Th1)-, Th2-, or Th17-associated immune response. Regardless of the adjuvant used, Nlrp10-/- mice failed to mount a T cell-dependent response, which was subsequently found to be caused by a lack of T cell priming. This defect was not attributable to an intrinsic problem in Nlrp10-/- T cells as it was shown that these cells could be differentiated in vitro into Th1, Th2, and Th17 cells, and that T cell receptor transgenic, Nlrp10-/- OT-II T cells could proliferate in wild-type mice following immunization. It also did not seem to be caused by a defect in Nlrp10-/- bone marrow-derived dendritic cells (BMDCs) since Nlrp10-/- BMDCs expressed the expected levels of MHC class II and CD86, were capable of efficient phagocytosis, and could activate naïve T cells in vitro. Despite these seemingly normal characteristics, antigen-loaded Nlrp10-/- BMDCs were unable to activate naïve OT-II T cells following adoptive transfer into wild-type mice, indicating that BMDC-expressed Nlrp10 was required for T cell activation in vivo.
To determine whether Nlrp10-/- dendritic cells (DCs) could interact with naïve T cells following antigen stimulation, fluorescein isothiocyanate (FITC) skin painting experiments were used to visualize DC migration from the skin to the lymph nodes.25 Using this technique, Nlrp10-/- mice were found to contain fewer DCs in the draining lymph nodes when compared to wild-type mice, both in the absence and in the presence of antigen. Notably, FITC-labeled Nlrp10-/- DCs were detected at the site of FITC application in the ear, confirming that Nlrp10-/- DCs were viable. Additionally, the authors demonstrated that when Nlrp10-/- mice were exposed to an inhalable, FITC-labeled, non-soluble antigen, the antigen could be detected in the lungs eighteen hours later, but not in the lung draining lymph nodes. Together these observations suggested that the defect in T cell activation in Nlrp10-/- mice was caused by an inability of migratory DCs to transport antigen from the site of infection to the draining lymph nodes where antigen presentation and CD4+ T cell differentiation takes place.
To investigate whether the reduction in DC migration to the lymph nodes was caused by an intrinsic or extrinsic defect in Nlrp10-/- DCs, the migration of labeled, activated Nlrp10-/- and wild-type BMDCs was monitored following co-injection into Nlrp10-deficient or wild-type mice.25 In both cases, the number of Nlrp10-/- DCs detected in the lymph nodes was considerably lower than the number of wild-type DCs detected there eighteen hours after infection, leading to the conclusion that Nlrp10-/- DCs have an intrinsic migratory defect. Significantly, the DC populations detected in the non-lymphoid tissues of naïve wild-type and Nlrp10-/- mice were equivalent, confirming that Nlrp10-/- migratory DCs developed normally and were capable of properly migrating to non-lymphoid peripheral tissues. In addition, it was shown that Ccr7 expression was normal in LPS-stimulated Nlrp10-/- DCs and that these cells could migrate toward CCR7 ligands, CXCL12, and Sphingosine-1-Phosphate in vitro. This was important because it indicated that Nlrp10-/- DCs could respond to chemoattractant signals and demonstrated that the migratory defect caused by the loss of Nlrp10 was mechanistically different than the chemokine homing defect previously observed in Ccr7- or Nlrp12-deficient DCs. 28, 29 Subsequent experiments using two photon laser scanning microscopy allowed the authors to visualize the movement of individual fluorescently-labeled Nlrp10-/- and wild-type DCs in vivo following co-injection into wild-type mice.25 While wild-type DCs migrated away from the injection site, Nlrp10-/- DCs did not, confirming the conclusion that Nlrp10 is necessary for DC migration from inflamed tissue to the draining lymph nodes where CD4+ T cell activation occurs.
The requirement of Nlrp10 for initiation of the adaptive immune response was further supported by a second report, which investigated the immune response to Candida albicans in Nlrp10-/- mice.26 In this study, Joly et al. found that Nlrp10-deficient mice were highly susceptible to C. albicans infection relative to wild-type mice. Nlrp10-/- mice displayed significant kidney damage, diminished renal function, and higher kidney fungal burdens than wild-type mice nine days after infection with C. albicans, and by day sixteen, 100% of the Nlrp10-/- mice had died compared to only 20% of wild-type mice. These results were reminiscent of those obtained with C. albicans-infected Nlrp3-deficient mice and suggested that like Nlrp3, Nlrp10 is required for anti-fungal immunity.30, 31 Subsequent experiments using bone marrow chimeric mice demonstrated that the increased susceptibility of Nlrp10-/- mice to C. albicans infection was primarily due to the loss of Nlrp10 in hematopoietic cells.
To further investigate the cause of the reduced immune response to C. albicans in Nlrp10-/- mice, the authors asked whether the loss of Nlrp10 affected inflammasome activation or the expression of other pro-inflammatory cytokines.26 Consistent with the previous report by Eisenbarth et al., both Nlrp10-/- and wild-type bone marrow-derived macrophages were found to secrete similar amounts of IL-1 beta following stimulation with C. albicans or other Nlrp3, Nlrc4, or AIM2 inflammasome agonists. In addition, Nlrp10-/- macrophages secreted approximately the same levels of IL-6, TNF-alpha, and IL-12 p40 as wild-type macrophages following treatment with lipopolysaccharide. As a result, Joly et al. concluded that Nlrp10 is not required for inflammasome-dependent or inflammasome-independent innate pro-inflammatory cytokine production.
While the loss of Nlrp10 did not affect cytokine secretion, there was a significant reduction in the Th1 and Th17 immune responses in Nlrp10-/- mice following C. albicans infection.26 This defect in the adaptive immune response was revealed by measuring the secretion of IFN-gamma and IL-17 from restimulated splenocytes removed from wild-type or Nlrp10-deficient mice fourteen days after infection. The results showed that splenocytes from C. albicans-infected Nlrp10-/- mice secreted significantly lower levels of IFN-gamma and IL-17 than splenocytes from wild-type mice, supporting the conclusion that Nlrp10 is required for initiation of the T cell-dependent adaptive immune response.25, 26 To determine if the reduced Th1/Th17-mediated immune response was responsible for the increased susceptibility of Nlrp10-/- mice to C. albicans infection, naïve CD4+ T cells or CD4+ T cells that had been previously challenged with a sublethal dose of C. albicans were adoptively transferred into Nlrp10-deficient mice followed by infection with a lethal dose of C. albicans.26 As expected, mice that received the previously challenged CD4+ T cells had a significantly higher survival rate than those that received naïve CD4+ T cells. This result strongly suggested that Nlrp10 plays an essential role in T cell activation, which is necessary to mount an appropriate immune response against C. albicans.
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Nlrp10 and Nlrp3 are Required for the Immune Response to Candida albicans Infection. Following infection by C. albicans, Nlrp3 and Nlrp10 are required to mount a protective anti-fungal immune response. Nlrp3 is essential for assembly of the Nlrp3 inflammasome, Caspase-1 activation, and the subsequent secretion of the innate, pro-inflammatory cytokines, IL-1 beta and IL-18 by bone marrow-derived macrophages and dendritic cells. In contrast, Nlrp10 has an inflammasome-independent function that is required for initiation of the T cell-dependent adaptive immune response. Nlrp10 was shown to be essential for bone marrow-derived dendritic cells (BMDCs) to migrate from the site of infection to the draining lymph nodes where antigen presentation and CD4+ T cell activation occurs. Significantly, this function of Nlrp10 was mechanistically independent of the ability of Nlrp10-/- BMDCs to respond to chemokine signals, and was required for the immune response not only to C. albicans, but also following immunization with multiple other adjuvants. These observations suggest that Nlrp10 has a novel role in globally regulating dendritic cell (DC)-dependent antigen transport and the initiation of the adaptive immune response.
Contrary to its original characterization, the studies by Eisenbarth et al. and Joly et al. suggest that Nlrp10 is a pro-inflammatory NLR with a unique involvement in initiation of the adaptive immune response.25, 26 Rather than regulating activation of the inflammasome and pro-inflammatory cytokine secretion, Nlrp10 is required for DC migration from the site of the infection to the draining lymph nodes where CD4+ T cell priming can occur.25 As a result, Nlrp10 provides an essential, inflammasome-independent link between the innate and adaptive immune response. Further research is necessary to determine the mechanism by which Nlrp10 regulates DC migration and to understand how differences in the activity of Nlrp10 in different cell types may contribute to the immune response.
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