NLRP3 inflammasome expression in cultured hepatocytes is regulated by nuclear factor-κB (NF-κB)

Background: The inflammasomes are cytoplasmic multiprotein complexes that are responsible for activation of inflammatory reactions. In principal, there are four individual inflammasome branches (i.e. NLRP1, NLRP3, NLRC4/NALP4, and AIM2) that mediate the cleavage and activation of caspase-1 and IL-1β that in turn leads to a complex network of cellular reactions that at the end initiate local and systemic inflammatory reactions [1]. We have recently shown that NLRP3 is virtually absent in cultured hepatocytes and that in vitro the stimulation of hepatocytes with lipopolysaccharides (LPS) results in strong activation of NLRP3 expression [2]. In this study we investigated the impact of NF-κB signalling on NLRP3 activation in primary hepatocytes.

Methods: Murine primary hepatocytes were isolated after the collagenase method established by Seglen [3]. Cells were plated on collagen-coated plates. The NF-κB activation inhibitor QNZ (N4-[2-(4-phenoxyphenyl)ethyl]-4,6-quinazolinediamine) and the adenoviral expression vector Ad5-CMV-IκB(S32A/S36A) expressing a hemagglutinin-tagged human superrepressor of NF-κB [4] were used to block NF-κB signalling. After LPS challenge, the expression of TNF-α, IL-1β and NLRP3 was analysed by qRT-PCR, Western blot and immunohistochemistry. The influence of NF-κB signalling on NLRP3 expression was further investigated in a novel murine hepatocytic cell system allowing directed depletion of the NF-κB essential modulator (NEMO).

Results: We found that QNZ blocks NF-κB-dependent expression of TNF-α, IL-1β and NLRP3. Likewise, the superrepressor of NF-κB prevents expression of NLRP3 and significantly reduces expression of inflammatory marker genes in liver cells. In cells that were depleted for NEMO, we could confirm that there is a close link of NF- κB activity and NLRP3 expression suggesting that the NF-κB pathway is the major pathway that controls NLRP3 expression.

Conclusions: We conclude that NF-κB signalling is a necessary prerequisite for proper activation of the NLRP3 inflammasome in primary hepatocytes. Our data further suggests that targeting NF-κB will be therapeutic useful to interfere with NLRP3 inflammasome activation.

References cited:

[1] Martinon F, Mayor A, Tschopp J. The inflammasomes: guardians of the body. Annu. Rev. Immunol. 2009;27:229 – 265.

[2] Boaru SG, Borkham-Kamphorst E, Tihaa L, Haas U, Weiskirchen R. Expression analysis of inflammasomes in experimental models of inflammatory and fibrotic liver disease. J. Inflamm. (Lond). 2012;9(1):49.

[3] Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29 – 83.

[4] Iimuro Y, Nishiura T, Hellerbrand C, Behrns KE, Schoonhoven R, Grisham JW, Brenner DA. NFkappaB prevents apoptosis and liver dysfunction during liver regeneration. J Clin Invest. 1998;101:802 – 11.

Corresponding author: Weiskirchen, Ralf

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