NLRC3 inhibits MyD88-dependent activation of NF-κB
NLRC3 is expressed mainly by cells of the immune response44
, so to better understand its function, we focused on the innate immune pathway, specifically TLR signaling pathways. TLRs and MyD88 are important for the detection of bacterial and viral pathogen-associated molecular patterns and are often the first molecules to respond to infection. To initially assess the function of NLRC3, we transfected human embryonic kidney (HEK293T) cells with an NF-κB luciferase reporter vector, an activating protein and increasing concentrations of NLRC3. NLRC3 significantly inhibited NF-κB activation in a dose-dependent manner in response to an activating signal from MyD88 and TRAF6 and, to a lesser but not significant extent, through IRAK, but not through IKKα or p65 (). These results suggested that NLRC3 was able to interfere with upstream signaling via MyD88 and/or TRAF6 but not downstream activation by IKKα or p65.
Figure 1 NLRC3 inhibits MyD88- and TRAF6-dependent activation of NF-κB. (a) Luciferase activity in HEK293T cells transfected with a luciferase reporter vector driven by an NF-κB-responsive promoter, plus empty vector (EV) or vector encoding MyD88, (more ...)
As myeloid-monocytic cells are the main cell types that engage in TLR signaling, we assessed the ability of NLRC3 to inhibit MyD88-dependent activation of NF-κB in the human monocyte-like THP-1 cell line. We transduced THP-1 cells with recombinant retrovirus expressing a stable short hairpin RNA that targets mRNA encoding NLRC3 (shNLRC3) or overexpressing a vector encoding NLRC3. We found substantial expression of NLRC3
in human monocytic cell lines (THP-1 and U937) and of Nlrc3
in mouse primary bone marrow macrophages (Supplementary Fig. 1a
). We confirmed by real-time PCR the overexpression of NLRC3 and lower expression of mRNA encoding NLRC3 achieved through the use of shNLRC3 (Supplementary Fig. 1b
). To determine if NLRC3 was involved in TLR signaling, we stimulated those THP-1 lines with lipopolysaccharide (LPS). Overexpression of NLRC3 resulted in less TNF
mRNA, whereas shNLRC3 resulted in more TNF
mRNA (). In addition, the secretion of TNF and IL-6 was also inversely associated with the expression of NLRC3
mRNA (, ). These findings suggested that NLRC3 may be a negative regulator of NF-κB signaling in monocytes.
The transcription of genes encoding cytokines occurs after MyD88-TRAF6 signaling, followed by activation of the IKK complex and phosphorylation of the NF-κB inhibitor IκBα. This post-translational modification leads to the ubiquitination and subsequent degradation of IκBα. To assess if the differences observed in cytokine production were due to changes in IκBα modification, we assessed total and phosphorylated IκBα by immunoblot analysis. In cells overexpressing NLRC3, phosphorylation of IκBα was slightly delayed (). Conversely, cells with shNLRC3 had early phosphorylation of IκBα (). Together these results indicated that NLRC3 diminished LPS-dependent NF-κB signaling in a human monocytic cell line.
NLRC3 inhibits NF-κB by association with TRAF6
TRAF molecules are important intermediaries of the activation of NF-κB. Overexpression of NLRC3 was sufficient to inhibit the activation of NF-κB by TRAF6. To determine if NLRC3 was able to interact with TRAF6, we transiently transfected HEK293T cells to express V5-tagged TRAF6 (V5-TRAF6) and hemagglutinin-tagged NLRC3 (HA-NLRC3) and coimmunoprecipitated proteins from the cells; we found that NLRC3 associated with TRAF6 (). Because of the nature of overexpression systems and the lack of a high-quality antibody for the detection of endogenous NLRC3, we were unable to confirm that this interaction occurred with physiological amounts of NLRC3 or TRAF6. However, a search for TRAF-binding domains in NLRC3 showed that human NLRC3 has two major TRAF2-binding motifs ((P/S/A/T)-X-(Q/E)-E, where ‘P/S/A/T’ indicates proline, serine, alanine or threonine, ‘X’ indicates any amino acid, and ‘Q/E’ indicates glutamine or glutamic acid) in its NBD (). However, it has been noted that the TRAF2-binding domain is not specific to TRAF2 but is indicative of a general TRAF-binding motif45
. For example, Nod2 has a predicted TRAF2-binding motif in its nucleotide-binding oligomerization domain, which facilitates association with TRAF4 (ref. 46
). We found that the region in NLRC3 with the TRAF motif is conserved across various species (). Because of homology in the NLR family, we searched the sequences of other NLR proteins for TRAF-binding motifs. We found that most NLR proteins have a major TRAF2-binding sequence, a minor TRAF2-binding motif and/or a TRAF6-binding motif, as described before47
(). These data indicated that interactions between NLR and TRAF molecules may be prevalent and identified previously unknown potential intersections of NLR proteins with TRAF proteins.
NLRC3 inhibits NF-κB by association with TRAF6.
Minor and major TRAF2-binding motifs and TRAF6-binding motifs in NLR proteins
We next assessed the possibility that NLRC3 associates with TRAF6 through that TRAF-binding motif in the NBD. We substituted alanine for all four amino acids in either the TRAF-binding site with the sequence Ser-Leu-Gln-Glu (site 1) or the TRAF-binding site with the sequence Ser-Val-Glu-Glu (site 2). We expressed those NLRC3 mutants together with V5-TRAF6 in HEK293T cells and assessed the association of TRAF6 and NLRC3 by immunoprecipitation of V5-TRAF6 and immunoblot analysis of HA-NLRC3. The substitution in site 1 of NLRC3 resulted in much less association, whereas the substitution in site 2 had less or little effect (). These findings indicated that the first TRAF-binding motif in the NBD of NLRC3 was important for its association with TRAF6.
NLRC3 influences the degradation and ubiquitination of TRAF6
We next explored the mechanism by which NLRC3 affected the function of TRAF6. When we transiently transfected HEK293T cells to express V5-TRAF6 and/or HA-NLRC3, we consistently noted less V5-TRAF6 in the presence of HA-NLRC3, whereas the amount of TRAF2 or TRAF3 (control proteins) remained unchanged (). Additionally, the NLRC3-dependent decrease in TRAF6 protein was dose dependent (). Proteins are frequently targeted to the proteosome for degradation by conjugation with K48-linked ubiquitin chains, whereas K63-linked ubiquitin is a common activating signal. Other NLR proteins that regulate NF-κB, such as Nod1 and Nod2, do so through interactions with ubiquitinated substrates, such as RIP2 (refs. 13, 14). To determine if NLRC3 affected the ubiquitination of TRAF molecules, we transfected HEK293T cells to express Myc-tagged TRAF2 or TRAF6, as well as hemagglutinin-tagged ubiquitin, in the presence or absence of vector encoding Flag-tagged NLRC3. We lysed cells in a stringent 1% SDS buffer and boiled them so that only covalent modifications remained. We immunoprecipitated TRAF proteins and quantified ubiquitin by densitometry. The presence of NLRC3 did not significantly affect the amount of ubiquitin on TRAF2 (, ) but did result in less ubiquitination of TRAF6 (, ). To determine the type of ubiquitin on TRAF6 that was affected by NLRC3, we transfected HEK293T cells to express TRAF6 with NLRC3 in the presence of plasmid encoding total ubiquitin, K48-linked ubiquitin or K63-linked ubiquitin. The presence of NLRC3 resulted in less total ubiquitin on TRAF6, accompanied by less K63-linked ubiquitin, but there was no significant change in K48-linked ubiquitin (, ). Although there was an apparent trend by which the presence of NLRC3 resulted in more K48-linked ubiquitination of TRAF6, the difference was variable and was not significant. This indicated that NLRC3 diminished the activating K63-linked ubiquitination of TRAF6 but left unchanged or enhanced the degradative K48-linked ubiquitination. The finding that K48-linked ubiquitin on TRAF6 remained in the presence of NLRC3 was consistent with the overall lower abundance of TRAF6 in the presence of NLRC3. Additionally, if NLRC3 prevented the accumulation of K63-linked ubiquitin on TRAF6 and left only K48-linked ubiquitin, then TRAF6 may have not only remained inactive but also may have been increasingly targeted to the proteosome. Because tight regulation of TRAF6 is important for NF-κB signaling, NLRC3 may inhibit proinflammatory signaling through this modification.
Figure 3 NLRC3 promotes degradation of TRAF6 and prevents autoubiquitination of TRAF6. (a) Immunoblot analysis of HEK293T cells transfected to overexpress vectors encoding Myc-tagged TRAF2 or V5-tagged TRAF3 or TRAF6 with or without Flag- or HA-tagged NLRC3. ( (more ...)
More proinflammatory cytokines made by Nlrc3−/− macrophages
The studies reported above relied on overexpressed proteins, which are useful for determining mechanistic events but can provide results that are inconsistent with endogenous processes. Therefore, to address the physiological role of NLRC3 in vivo
, we generated Nlrc3
) mice by homologous recombination (). The NBD, which is encoded by exon 3, is important for NLR protein function; therefore, we designed a targeting vector that, after homologous recombination with the endogenous locus, removed exon 3, replacing it with the gene encoding resistance to the aminoglycoside antibiotic geneticin. We used embryonic stem cells from 129S6 mice that expressed the properly modified locus to generate chimeric mice. We bred male chimeras with female C57BL/6 mice and identified germline transmission in the offspring by coat color and confirmed this by Southern blot analysis (). We bred those offspring to C57BL/6J mice. We intercrossed heterozygous (Nlrc3+/−
) mice for nine generations to generate the F9 Nlrc3−/−
mice used in these studies. Mendelian ratios for each genotype and the distribution of the targeted allele among male and female mice were normal. We verified genotype by PCR and determined absence of expression by RT-PCR (Supplementary Fig. 2a
mice did not seem to have any anatomic abnormalities, similar to mice lacking other NLR proteins. Reproductive performance, gender distribution, frequency of lymphocytes and monocytes in the spleen, and total number of splenocytes, peritoneal cells and bone marrow cells were equivalent to those of wild-type mice (Supplementary Fig. 2b
Figure 4 Nlrc3−/− macrophages produce more proinflammatory cytokines in response to TLR agonists. (a) Strategy used to disrupt the Nlrc3 locus: homologous recombination of the targeting construct (middle) with endogenous wild-type (WT) Nlrc3 (top) (more ...)
As NLRC3 resulted in less production of proinflammatory cytokines in human cell lines (), we sought to assess if a change in cytokines also occurred in Nlrc3−/−
macrophages treated with LPS and whether this change was due to a greater abundance of transcripts encoding proinflammatory cytokines. The cytokine-encoding genes Tnf
are targets for activation by NF-κB, and expression of these genes increases considerably after stimulation of the TLR. To determine if Nlrc3−/−
macrophages had higher expression of genes encoding proinflammatory cytokines, we measured transcripts by real-time PCR. There was much higher expression of those cytokine-encoding genes in Nlrc3−/−
peritoneal macrophages than in wild-type cells after 1 h of stimulation with LPS (). However, there was no difference between wild-type and Nlrc3−/−
cells in expression of the gene encoding interferon-β after stimulation with LPS (). At later time points, the expression of Tnf, Il1b
cells was similar to that in wildtype cells. Differences in the expression of TLR or TLR-signaling molecules could lead to variation in cytokine release. However, the expression of Tlr4
in peritoneal macrophages was similar in wild-type and Nlrc3−/−
cells (). Additionally, a negative feedback loop can be activated through NF-κB, by which expression of NF-κB inhibitors such as A20, CYLD and IκBα is increased. To assess this possible repressive effect, we measured expression of the genes encoding A20 (Tnfaip3
; called ‘A20
’ here), CYLD (Cyld
) and IκBα (Nfkbia
) before and after stimulation with LPS. We observed no difference between wild-type and Nlrc3−/−
macrophages in their expression of Cyld
before or after stimulation (). In contrast, A20
expression was modestly higher in Nlrc3−/−
macrophages than in wild-type macrophages at 1 h after stimulation with LPS and was much higher in Nlrc3−/−
macrophages than in wild-type macrophages by 6 h after treatment with LPS (). That finding was in agreement with published reports indicating a negative regulatory loop between activation of NF-κB and A20
. These data indicated that the absence of NLRC3 resulted in an early increase in the transcription of NF-κB-dependent genes activated by TLR4. That change was not preceded by a change in the expression of Tlr4, Traf6, Cyld
Peritoneal macrophages from Nlrc3−/− mice also secreted more TNF and IL-6 than did wild-type macrophages in response to TLR agonists that activate signaling via the MyD88-dependent pathway (the synthetic lipopeptide Pam3Cys for TLR2; LPS for TLR4; and single-stranded RNA for TLR7 and TLR8) but not to a significant extent in response to an agonist that activates signaling via the TRIF-dependent pathway (the synthetic RNA duplex poly(I:C) for TLR3; ). Additionally, quantification of TRAF6 protein in wild-type and Nlrc3−/− peritoneal macrophages showed that cells lacking NLRC3 had more TRAF6 (). Bone marrow–derived macrophages also had more TRAF6, although this greater abundance was not as substantial and varied by experiment (data not shown). That observation supported the earlier finding obtained with HEK293T cells showing that overexpression of NLRC3 led to less TRAF6 protein. To determine if Nlrc3 expression was affected by stimulation of the TLR, we measured Nlrc3 expression in macrophages after in vitro stimulation with LPS. Expression diminished soon after stimulation and remained low throughout the assay interval, although by the end of the study, there was a modest recovery (). As the macrophage culture began to lose viability at those later time points, the lack of recovery of Nlrc3 expression might have been to due to the unhealthy state of the culture. To overcome the artifacts of tissue culture, we examined the in vivo effect of LPS on Nlrc3 expression in peritoneal cells isolated at various time points after intraperitoneal injection of LPS. Nlrc3 expression decreased after stimulation and this decrease was sustained for 48 h, but by 108 h, Nlrc3 expression recovered to the amount before stimulation (). That finding suggested that the decrease in Nlrc3 expression during LPS treatment was probably necessary for the inflammatory response to proceed, whereas its recovery later restored the basal resting state (). Thus, regulation of Nlrc3 mRNA expression provides a mechanism by which NLRC3 function is controlled.
Figure 5 Nlrc3−/− macrophages produce more proinflammatory cytokines and TRAF6 in response to LPS. (a) Production of TNF and IL-6 by wild-type and Nlrc3−/− peritoneal macrophages left untreated (UT) or stimulated via the TLR. ssRNA, (more ...)
Because there was less TRAF6 protein in the presence of NLRC3, whereas LPS diminished Nlrc3 expression, we assessed the effect of NLRC3 on TRAF6 during stimulation with LPS. We measured TRAF6 protein in wild-type and Nlrc3−/− peritoneal macrophages before and after stimulation with LPS. In basal conditions, Nlrc3−/− macrophages had much more TRAF6 than did wild-type cells. However, at 24 h after LPS stimulation, when Nlrc3 expression was low in wild-type cells, wild-type and Nlrc3−/− cells had an equal amount of TRAF6 ().
More activation of TRAF6 and NF-κB in Nlrc3−/− macrophages
To better determine the function of NLRC3 in NF-κB signaling, we explored the mechanism by which NLRC3 inhibited the macrophage proinflammatory response. Because we had observed less ubiquitinated TRAF6 in the presence of NLRC3 when those proteins were overexpressed, we assessed if the deletion of Nlrc3 resulted in more ubiquitination of TRAF6. To measure ubiquitination, we activated TRAF6 by timed stimulation of bone marrow–derived macrophages with LPS. We immunoprecipitated endogenous TRAF6 and measured K63-linked ubiquitin associated with TRAF6 through the use of an antibody specific for K63-linked ubiquitin chains. We did not study K48-linked ubiquitin because antibodies to K48-linked ubiquitin did not work properly in our experiments. We used stringent lysis conditions to disrupt any noncovalent bonds. There was more K63-linked ubiquitin on TRAF6 from Nlrc3−/− cells than on TRAF6 from wild-type control cells at all time points, and possibly even in the absence of stimulation (). These data suggested that NLRC3 negatively regulated K63-linked ubiquitination of TRAF6, which would be expected to diminish downstream signaling. To determine if the absence of NLRC3 led to more NF-κB signaling, we assessed the nuclear localization of p65 and the abundance of p65 and IκBα. After stimulation with LPS, the nuclei of Nlrc3−/− macrophages had much more p65 than did those of wild-type cells (); nearly 70% more nuclei contained p65 in Nlrc3−/− macrophages than in wild-type cells (). Additionally, there was earlier and more-prolonged phosphorylation of p65 in Nlrc3−/− macrophages than in wild-type cells (). That greater phosphorylation correlated with more-rapid loss of total IκBα and lack of recovery of IκBα at 60 min (). TRAF6 can affect other proinflammatory pathways, notably the mitogen-activated protein kinase signaling pathway. To determine if the effect of NLRC3 on TRAF6 was specific for the activation of NF-κB, we examined phosphorylation of the mitogen-activated protein kinases p38, Erk and Jnk after stimulation with LPS. Signaling via this pathway was not affected much in the absence of NLRC3 ().
Figure 6 Nlrc3−/− macrophages have more K63-linked ubiquitination of TRAF6 and activation of NF-κB. (a) Immunoblot analysis of K63-linked ubiquitination (K63-Ub) of endogenous TRAF6 immunoprecipitated from wild-type and Nlrc3−/− (more ...)
Enhanced response of Nlrc3−/− mice to endotoxic shock
The experiments reported above indicated that NLRC3 functioned as an attenuator of the response to LPS in macrophages. We next assessed its role as an in vivo regulator of the severe inflammatory condition of endotoxic shock. LPS is a common component of Gram-negative bacteria and induces the production of proinflammatory cytokines. An uncontrolled immune response to LPS can lead to a cytokine storm and serves as a surrogate model of sepsis. The NF-κB pathway is a chief driver of this inflammation. Because of the enhanced release of proinflammatory cytokines in response to TLR agonists in both human and mouse cells lacking NLRC3, we hypothesized that Nlrc3−/− mice would be more susceptible to LPS-induced shock. To assess this, we injected wild-type and Nlrc3−/− mice with LPS or PBS (vehicle control) and monitored temperature and weight. In response to a nonlethal dose of LPS, wild-type and Nlrc3−/− mice initially had a similar drop in temperature and weight. However, Nlrc3−/− mice had a significant delay in recovery, as measured by their lower body temperature and weight after challenge with LPS (, ).
Figure 7 Nlrc3−/− mice have a more severe reaction to sublethal challenge with LPS than do wild-type mice. (a, b) Change in body temperature (a) and weight (b) over time in wild-type and Nlrc3−/− mice challenged with LPS (4.5 mg (more ...)
The body temperatures of wild-type and Nlrc3−/− mice began to diverge 18–42 h after LPS challenge, with Nlrc3−/− mice showing a greater drop than that of control mice. Similarly, Nlrc3−/− mice lost more weight than control mice did from 42 h to 88 h after LPS stimulation. To determine a possible cause for the diminished health of Nlrc3−/− mice, we killed the mice at 24 h after the LPS challenge. Because we injected the mice intraperitoneally, we collected peritoneal lavage fluid to assess not only cellularity due to inflammatory infiltrates but also local cytokine concentrations, and we collected serum to assess systemic cytokine concentrations. The serum of Nlrc3−/− mice had a significantly higher concentration of IL-6 at 24 h after LPS treatment than did wild-type serum; additionally, the concentration of TNF was also higher in Nlrc3−/− serum than in wild-type serum (). We noted a much greater abundance of Il6 transcripts and IL-6 protein in the peritoneal cavity of Nlrc3−/− mice than in that of wild-type mice at 24 h after LPS challenge (, ). By 48 h, the concentration of IL-6 was lower in both wild-type and Nlrc3−/− mice (, ). Additionally, Nlrc3−/− peritoneal cells had higher expression of genes encoding the proinflammatory cytokines IL-6 and TNF than did wild-type peritoneal cells (). The absence of NLRC3 affected TNF less than it affected IL-6 and did not have a significant effect on IL-1β (–). As Nlrc3−/− peritoneal macrophages produced more proinflammatory cytokines ex vivo, it is possible that the enhancement in systemic cytokines was due to changes in cellular composition. To assess this, we examined the cellular composition of the peritoneal cavity of wild-type and Nlrc3−/− mice after challenge with LPS. Although there was not a significant difference between the mice in the total proportion of cells in the peritoneal cavity, there was slightly greater frequency of peritoneal monocytes in Nlrc3−/− mice than in wild-type mice (). However, this modest difference was probably not responsible for greater differences in the abundance of Il6 transcripts and IL-6 protein. Together these results indicated that NLRC3 had a protective role in endotoxic shock by diminishing proinflammatory cytokines, especially IL-6 and, to a lesser extent, TNF. In contrast to inflammasome NLR proteins, NLRC3 had little effect on IL-1β. The lack of an effect on IL-1β might have accounted for the relatively modest effect of NLRC3 on body temperature during exposure to endotoxins.