Cellular Microbiology (2012) doi:10.1111/j.1462-5822.2012.01822.x NLRP10 enhances Shigella-induced pro-inflammatory responses Katja Lautz,1 Anna Damm,1 Maureen Menning,1 Introduction Julia Wenger,2 Alexander C. Adam,3 Paola Zigrino,4 Elisabeth Kremmer5 and Thomas A. Kufer1* The innate immune system is the first line of defence 1Institute for Medical Microbiology, Immunology and against invading pathogens in mammals. By gaining Hygiene, University of Cologne, Cologne, Germany. access to the cellular cytoplasm, some bacteria and 2Department of Molecular Biology, University of viruses adopted to escape host innate immune surveil- Salzburg, Salzburg, Austria. lance by membrane standing pattern-recognition recep- 3Department of Pathology, University of Cologne, tors (PRRs) such as the Toll-like receptor family. To cope Cologne, Germany. with such intracellular residing pathogens, the host has 4Department of Dermatology, University of Cologne, evolved sophisticated detection systems. In particular Cologne, Germany. members of the nucleotide-binding domain leucine-rich 5Helmholtz Zentrum München, Institute of Molecular repeat containing family (NLR) have been shown to sense Immunology, Munich, Germany. such invasive bacteria and viruses. NLRs are a heteroge- neous class of 23 AAA+ ATPases in humans, which are characterized by a tripartite structural organization com- Summary prising an effector domain, a STAND type ATPase domain Members of the NLR family evolved as intracellular (called NACHT domain) and a series of leucine-rich sensors for bacterial and viral infection. However, repeats (LRRs) at the carboxyl-terminus (Fritz et al., our knowledge on the implication of most of the 2006; Schroder and Tschopp, 2010). NLRs are classified human NLR proteins in innate immune responses based on the nature of their amino-terminal effector still remains fragmentary. Here we characterized domain into a CARD-domain (NLRC) and a PYD-domain the role of human NLRP10 in bacterial infection. Our subfamily (NLRP), as well as in an ‘atypical’ effector data revealed that NLRP10 is a cytoplasmic local- domain family, comprising CIITA, NAIP, NLRX1, NLRC3 ized protein that positively contributes to innate and NLRC5. Most of our current knowledge of the biology immune responses induced by the invasive bacte- of NLRs derives from studies on a small number of NLRs rial pathogen Shigella flexneri. SiRNA-mediated including NOD1, NOD2 and NLRC4 as well as NLRP3 knock-down studies showed that NLRP10 contrib- and NLRP1 (Fritz et al., 2006, Schroder and Tschopp, utes to pro-inflammatory cytokine release triggered 2010). Seminal work has shown that NOD1 and NOD2 by Shigella in epithelial cells and primary dermal are sensors for bacterial infection and mediate inflam- fibroblasts, by influencing p38 and NF-kB activa- matory responses whereas NLRP1 and NLRP3 can form tion. This effect is dependent on the ATPase activity multimeric protein complexes, the so-called inflammas- of NLRP10 and its PYD domain. Mechanistically, omes, which allow processing of caspase-1 and subse- NLRP10 interacts with NOD1, a NLR that is pivotally quent release of IL-1b and IL-18 after sensing of PAMPs involved in sensing of invasive microbes, and both or danger signals (Martinon et al., 2002; Inohara et al., proteins are recruited to the bacterial entry point 2003; Girardin et al., 2003a; 2003b). However, the contri- at the plasma membrane. Moreover, NLRP10 phy- bution of other NLRs in bacteria-mediated innate immune sically interacts with downstream components responses remains less well defined. of the NOD1 signalling pathway, such as RIP2, Here we used the Gram-negative, invasive bacte- TAK1 and NEMO. Taken together, our data revealed rium Shigella flexneri as a well-characterized model a novel role of NLRP10 in innate immune re- system for bacterial infection. S. flexneri is a human sponses towards bacterial infection and suggest pathogen, responsible for endemic dysentery and dis- that NLRP10 functions as a scaffold for the forma- plays a complex interplay with the host cell (Schroeder tion of the NOD1–Nodosome. and Hilbi, 2008). S. flexneri has been shown to trigger pro-inflammatory responses in human epithelial cells by activating the NLR proteins NOD1 and NOD2 (Girardin Received 15 March, 2012; revised 18 May, 2012; accepted 26 May, 2012. *For correspondence. E-mail [email protected]; et al., 2001; Kufer et al., 2006). In macrophages NLRC4, Tel. (+49) 221 478 7279; Fax (+49) 221 478 7288. NAIP and NLRP3 contribute to Shigella-mediated host © 2012 Blackwell Publishing Ltd cellular microbiology 2 K. Lautz et al. responses as well by inducing a special type of caspase- NLRP10 and involves physical interactions with NOD1 1-dependent cell death (Damiano et al., 2004; Suzuki and the NOD1 signalling pathway components NEMO, et al., 2007; Willingham et al., 2007). The role of further RIP2 and TAK1. Conclusively, this defines a novel func- NLRs in Shigella-induced innate immune responses tion of NLRP10 in innate immune responses towards bac- however remains largely elusive. Of particular interest in terial infection and suggests that NLRP10 modulates our studies was NLRP10, the smallest of all human NLRs. signalling induced by the Nodosome complex. NLRP10 (Nalp10, alternatively named PAN5; NOD8; PYNOD; CLR11.1) is a prototypic member of the NLR family, although it lacks the LRRs (Ting et al., 2008), Results which are proposed to mediate detection of PAMPs. A NLRP10 is a cytoplasmic protein expressed in epithelial regulatory role for NLRP10 in innate immune responses and fibroblastic cells thus seems more likely than its function as a PRR. NLRP10 expression was proposed to be induced by In order to identify a suitable cellular model system for the PAMPs, such as LPS (Wang et al., 2004) and it was detailed functional characterization of NLRP10, we ana- shown that NLRP10 can inhibit ASC-mediated NF-kB acti- lysed the expression pattern of human NLRP10. While vation and IL-1b release in epithelial cells (Wang et al., human NLRP10 mRNA was shown to be expressed 2004; Kinoshita et al., 2005; Imamura et al., 2010). highly in heart, brain and muscle (Wang et al., 2004), Accordingly, transgenic mice that overexpress NLRP10 another study found that NLRP10 expression is highest in show a reduced IL-1b response towards bacterial and liver, small intestine and muscle (Lech et al., 2010). In PAMP stimulation and are less susceptible for endotoxic accordance with lack of detectable expression of NLRP10 shock (Imamura et al., 2010). These findings emphasize a in primary human CD14+ cells (Lech et al., 2010) we function of NLRP10 in innate immune responses and found that NLRP10 mRNA was not robustly detectable in suggest a role of NLRP10 in regulating inflammasome- the human myeloid cell line THP1 even after differentia- mediated responses. A physiological role for NLRP10 in tion to macrophage-like cells by PMA. Although NLRP10 immunity has been put forward by the characterization expression was slightly induced after additional stimula- of NLRP10-deficient mice. Surprisingly, these animals tion with LPS (Fig. 1A). Still, primary human peripheral display a migration defect of activated dendritic cells blood mononuclear cells (PBMCs) showed little expres- resulting in reduced adaptive immunity, whereas perito- sion of NLRP10 (Fig. 1A), as recently reported (Wang neal macrophages and bone marrow-derived dendritic et al., 2004). NLRP10 was found to be expressed in cells derived from NLRP10-deficient animals show normal human colon (Wang et al., 2004; Lech et al., 2010), we inflammasome activation (Eisenbarth et al., 2012). This therefore also tested NLRP10 mRNA expression in suggests that NLRP10 contributes to multiple cellular various human colon cell lines. This revealed that HT-29, functions. However, our understanding of the underlying CaCo2 and SW480 cells showed expression of NLRP10, molecular mechanisms remains largely elusive. although we found that epithelial HeLa cells had a much Here we show that NLRP10 contributes to pro- higher basal expression of NLRP10 (Fig. 1A). In contrast, inflammatory innate immune responses towards the inva- human embryonic kidney cells (HEK293T) showed no sive human pathogen S. flexneri. This effect is dependent detectable basal expression of NLPR10 transcript on a functional ATPase domain and the PYD domain of (Fig. 1A). Of note, primary human dermal fibroblasts Fig. 1. NLRP10 is a cytosolic protein predominantly expressed in human epidermis. A. Expression of NLRP10 mRNA in different human cell lines and primary human PBMCs, analysed by RT-PCR. Amplification of GAPDH served as control (lower panels). THP1 cells were incubated in 100 nM PMA for 24 h and exposed to LPS (100 ng ml-1) 4 h prior to the isolation of RNA where indicated. B. Expression of NLRP10 mRNA in human dermis and epidermis samples, tested by RT-PCR. N: negative control; P: plasmid positive control. Results are representative of three donors. C. Characterization of NLRP10-specific monoclonal antibody 4B4 in lysates from HEK293T transfected with NLRP10 or empty plasmid. Detection with a FLAG-specific antibody served as control. D. Immunohistochemical staining of healthy human skin tissue. Endogenous NLRP10 was detected with anti-NLRP10 4B4 (left panel). Control staining was conducted with a matched isotope (right panel). The result is representative of two donors. E. Indirect immunofluorescence micrographs of HeLa (left panel) and primary human dermal fibroblasts (right panel). Ectopically expressed FLAG-NLRP10 was detected by an anti-FLAG antibody (green), merge with staining for DNA (blue) is shown (left panel). Endogenous NLRP10 was detected with the 8H2 antibody (red) in primary human dermal fibroblasts. Merge with staining for DNA (blue) and actin (green) is shown. Data are representative for two donors. F. Immunoblot analysis of the primary epidermal fibroblasts treated for 72 h with an NLRP10-specific siRNA or a non-targeting control siRNA. Detection of endogenous NLRP10 in the same cells and with the same antibody used in (E) is shown. Probing for a-tubulin served as control for equal loading.