Genome Editing of Human Primary Keratinocytes by CRISPR/Cas9 Reveals an Essential Role of the NLRP1 Inflammasome in UVB Sensing

Abstract

By forming a protective barrier, epidermal keratinocytes represent the first line of defense against environmental insults. UVB radiation of the sun is a major challenge for the skin and can induce inflammation, aging, and eventually skin cancer. UVB induces an immune response in human keratinocytes resulting in activation and secretion of the proinflammatory cytokines proIL-1β and -18. This is mediated by an assembly of protein complexes, termed inflammasomes. However, the mechanisms underlying sensing of UVB by keratinocytes, and particularly the types of inflammasomes required for cytokine secretion, are a matter of debate. To address these questions, we established a protocol that allows the generation of CRISPR/Cas9-targeted human primary keratinocytes. Our experiments showed an essential role of the NLRP1 rather than the NLRP3 inflammasome in UVB sensing and subsequent IL-1β and -18 secretion by keratinocytes. Moreover, NLRP1 but not NLRP3 was required for inflammasome activation in response to nigericin, a potassium ionophore and well-established NLRP3 activator in immune cells. Because the CRISPR/Cas9-targeted cells retained their full differentiation capacity, genome editing of human primary keratinocytes might be useful for numerous research and medical applications.

Research Techniques Made Simple: CRISPR Genetic Screens

“…CRISPR screens can be performed in primary cells, such as keratinocytes, melanocytes, and fibroblasts (Fenini et al, 2018;Slivka et al, 2019;Sun et al, 2015). However, primary cells can be difficult to transfect, generate lower geneediting efficiency, or may have cell limitations that are incompatible with long-term library screens (Ford et al, 2019).…”

NLRP1 Is the Key Inflammasome in Primary Human Keratinocytes

“…Anthrax lethal toxin directly activates NLRP1 only in mice and rats, and the effect of Toxoplasma gondii on human NLRP1 is currently being investigated. Fenini et al (2018) introduce UVB and nigericin as ligands for NLRP1, and to some extent NLPR3, in human keratinocytes (Fenini et al, 2018). As previously shown, human keratinocytes secrete IL-1b, IL-18, and IL-1a in response to UVB treatment and nigericin (Feldmeyer et al, 2007;Gross et al, 2012;Yazdi et al, 2010).…”

“…Upon small interfering RNA knockdown of either NLRP3 or NLRP1 in human primary keratinocytes, IL-1b and IL-18 was diminished (Feldmeyer et al, 2007). Because NLRP1 was considered to be a key NLRP protein in keratinocytes (Zhong et al, 2016), Fenini et al (2018) used CRSPR/Cas9 technology to knock out genes in primary human keratinocytes.…”

“…Besides altered IL-1b secretion after UVB treatment, no differences in differentiation were observed. To determine which of the three inflammasomes were functionally relevant in keratinocytes (AIM2 [Dombrowski et al, 2011], NLRP3 [Feldmeyer et al, 2007], or NLRP1 [Feldmeyer et al, 2007;Zhong et al, 2016]), Fenini et al (2018) generated NLRP1 and NLRP3 sgRNA CRISPR/ Cas9-targeted human primary keratinocytes (Figure 1a). After priming with IFNg, NLRP3 was induced, while NLRP1, caspase-1, ASC, and pro-IL-1b were already present in untreated keratinocytes.…”

“…Because both UVB and nigericin are known to be toxic and both LDH release and IL-1 secretion depend on the presence of caspase-1, NLRP1, and ASC, NLRP1 and ASC might regulate pyroptosis or apoptosis. Fenini et al (2018) use sgRNA CRISPR/Cas9 targeting to knock down pattern recognition receptors in human primary keratinocytes. The exact mechanisms responsible for sensing of UVB or nigericin by NLRP1 remain to be elucidated.…”