Chlamydia evasion of neutrophil host defense results in NLRP3 dependent myeloid-mediated sterile inflammation through the purinergic P2X7 receptor.

Chlamydia trachomatis infection causes severe inflammatory disease resulting in blindness and infertility. The pathophysiology of these diseases remains elusive but myeloid cell-associated inflammation has been implicated. Here we show NLRP3 inflammasome activation is essential for driving a macrophage-associated endometritis resulting in infertility by using a female mouse genital tract chlamydial infection model. We find the chlamydial parasitophorous vacuole protein CT135 triggers NLRP3 inflammasome activation via TLR2/MyD88 signaling as a pathogenic strategy to evade neutrophil host defense. Paradoxically, a consequence of CT135 mediated neutrophil killing results in a submucosal macrophage-associated endometritis driven by ATP/P2X7R induced NLRP3 inflammasome activation. Importantly, macrophage-associated immunopathology occurs independent of macrophage infection. We show chlamydial infection of neutrophils and epithelial cells produce elevated levels of extracellular ATP. We propose this source of ATP serves as a DAMP to activate submucosal macrophage NLRP3 inflammasome that drive damaging immunopathology. These findings offer a paradigm of sterile inflammation in infectious disease pathogenesis.

CARD19, the protein formerly known as BinCARD, is a mitochondrial protein that does not regulate Bcl10-dependent NF-κB activation after TCR engagement.

After T cell receptor (TCR) engagement, the CARD11-Bcl10-Malt1 (CBM) complex oligomerizes to transduce NF-κB activating signals. Bcl10 is then degraded to limit NF-κB activation. The cDNA AK057716 (BinCARD-1) was reported to encode a novel CARD protein that interacts with Bcl10 and modestly inhibits NF-κB activation. In a later study, a second isoform, BinCARD-2, was identified. Here, we report that the cDNA AK057716 (BinCARD-1) is an incompletely spliced derivative of the gene product of C9orf89, whereas CARD19 (BinCARD-2) represents the properly spliced isoform, with conservation across diverse species. Immunoblotting revealed expression of CARD19 in T cells, but no evidence of BinCARD-1 expression, and microscopy demonstrated that endogenous CARD19 localizes to mitochondria. Although we confirmed that both BinCARD-1 and CARD19 can inhibit NF-κB activation and promote Bcl10 degradation when transiently overexpressed in HEK293T cells, loss of endogenous CARD19 expression had little effect on Bcl10-dependent NF-κB activation, activation of Malt1 protease function, or Bcl10 degradation after TCR engagement in primary murine CD8 T cells. Together, these data indicate that the only detectable translated product of C9orf89 is the mitochondrial protein CARD19, which does not play a discernible role in TCR-dependent, Bcl10-mediated signal transduction to Malt1 or NF-κB.

T cell receptor signals to NF-κB are transmitted by a cytosolic p62-Bcl10-Malt1-IKK signalosome.

Antigen-mediated stimulation of the T cell receptor (TCR) triggers activation of nuclear factor κB (NF-κB), a key transcriptional regulator of T cell proliferation and effector cell differentiation. TCR signaling to NF-κB requires both the Carma1-Bcl10-Malt1 (CBM) complex and the inhibitor of κB (IκB) kinase (IKK) complex; however, the molecular mechanisms connecting the CBM complex to activation of IKK are incompletely defined. We found that the active IKK complex is a component of a TCR-dependent cytosolic Bcl10-Malt1 signalosome containing the adaptor protein p62, which forms in effector T cells. Phosphorylated IκBα and NF-κB were transiently recruited to this signalosome before NF-κB translocated to the nucleus. Inhibiting the activity of the kinase TAK1 or IKK blocked the phosphorylation of IKK, but not the formation of p62-Bcl10-Malt1 clusters, suggesting that activation of IKK occurs after signalosome assembly. Furthermore, analysis of T cells from p62-deficient mice demonstrated that the p62-dependent clustering of signaling components stimulated activation of NF-κB in effector T cells. Thus, TCR-stimulated activation of NF-κB requires the assembly of cytosolic p62-Bcl10-Malt1-IKK signalosomes, which may ensure highly regulated activation of NF-κB in response to TCR engagement.

Selective autophagy of the adaptor protein Bcl10 modulates T cell receptor activation of NF-κB.

The adaptor protein Bcl10 is a critically important mediator of T cell receptor (TCR)-to-NF-κB signaling. Bcl10 degradation is a poorly understood biological phenomenon suggested to reduce TCR activation of NF-κB. Here we have shown that TCR engagement triggers the degradation of Bcl10 in primary effector T cells but not in naive T cells. TCR engagement promoted K63 polyubiquitination of Bcl10, causing Bcl10 association with the autophagy adaptor p62. Paradoxically, p62 binding was required for both Bcl10 signaling to NF-κB and gradual degradation of Bcl10 by autophagy. Bcl10 autophagy was highly selective, as shown by the fact that it spared Malt1, a direct Bcl10 binding partner. Blockade of Bcl10 autophagy enhanced TCR activation of NF-κB. Together, these data demonstrate that selective autophagy of Bcl10 is a pathway-intrinsic homeostatic mechanism that modulates TCR signaling to NF-κB in effector T cells. This homeostatic process may protect T cells from adverse consequences of unrestrained NF-κB activation, such as cellular senescence.

An active kinase domain is required for retention of PKCθ at the T cell immunological synapse.

Protein kinase Cθ (PKCθ) is a serine/threonine kinase that plays an essential role in antigen-regulated responses of T lymphocytes. Upon antigen stimulation, PKCθ is rapidly recruited to the immunological synapse (IS), the region of contact between the T cell and antigen-presenting cell. This behavior is unique among T cell PKC isoforms. To define domains of PKCθ required for retention at the IS, we generated deletion and point mutants of PKCθ. We used quantitative imaging analysis to assess IS retention of PKCθ mutants in antigen-stimulated T cell clones. Deletion of the kinase domain or site-directed mutation of a subset of known PKCθ phosphorylation sites abrogated or significantly reduced IS retention, respectively. IS retention did not correlate with phosphorylation of specific PKCθ residues but rather with kinase function. Thus PKCθ catalytic competence is essential for stable IS retention.

Multiple protein domains mediate interaction between Bcl10 and MALT1.

Bcl10 and MALT1 are essential mediators of NF-kappaB activation in response to the triggering of a diverse array of transmembrane receptors, including antigen receptors. Additionally, both proteins are translocation targets in MALT lymphoma. Thus, a detailed understanding of the interaction between these mediators is of considerable biological importance. Previous studies have indicated that a 13-amino acid region downstream of the Bcl10 caspase recruitment domain (CARD) is responsible for interacting with the immunoglobulin-like domains of MALT1. We now provide evidence that the death domain of MALT1 and the CARD of Bcl10 also contribute to Bcl10-MALT1 interactions. Although a direct interaction between the MALT1 death domain and Bcl10 cannot be detected via immunoprecipitation, FRET data strongly suggest that the death domain of MALT1 contributes significantly to the association between Bcl10 and MALT1 in T cells in vivo. Furthermore, analysis of point mutants of conserved residues of Bcl10 shows that the Bcl10 CARD is essential for interaction with the MALT1 N terminus. Mutations that disrupt proper folding of the Bcl10 CARD strongly impair Bcl10-MALT1 interactions. Molecular modeling and functional analyses of Bcl10 point mutants suggest that residues Asp(80) and Glu(84) of helix 5 of the Bcl10 CARD directly contact MALT1. Together, these data demonstrate that the association between Bcl10 and MALT1 involves a complex interaction between multiple protein domains. Moreover, the Bcl10-MALT1 interaction is the second reported example of interactions between a CARD and a non-CARD protein region, which suggests that many signaling cascades may utilize CARD interactions with non-CARD domains.