The NLRP12 Sensor Negatively Regulates Autoinflammatory Disease by Modulating Interleukin-4 Production in T Cells

Article

The NLRP12 Sensor Negatively Regulates Autoinflammatory Disease by Modulating Interleukin-4 Production in T Cells Highlights d

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Deficiency in NLRP12 promotes the generation of hyperinflammatory T cell responses Induction of EAE in Nlrp12 / mice results in atypical neuroinflammatory disease

Authors John R. Lukens, Prajwal Gurung, ..., Hongbo Chi, Thirumala-Devi Kanneganti

Correspondence

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NLRP12 is an intrinsic regulator of T cells

[email protected]

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Dysregulated IL-4 production causes T-cell-induced inflammation in Nlrp12 / mice

In Brief

Lukens et al., 2015, Immunity 42, 654–664 April 21, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.immuni.2015.03.006

Kanneganti et al. identify the NOD-like receptor protein, NLRP12, as a novel negative regulator of T cell activation. Their work defines a T-cell-autonomous role for NLRP12 in the suppression of inflammatory cytokine production and suggests that dysregulated NLRP12mediated signaling in T cells is sufficient to drive inflammatory disease.

Immunity

Article The NLRP12 Sensor Negatively Regulates Autoinflammatory Disease by Modulating Interleukin-4 Production in T Cells John R. Lukens,1,2 Prajwal Gurung,1 Patrick J. Shaw,1 Maggie J. Barr,1 Md. Hasan Zaki,3 Scott A. Brown,1 Peter Vogel,4 Hongbo Chi,1 and Thirumala-Devi Kanneganti1,* 1Department

of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA for Brain Immunology and Glia (BIG), Department of Neuroscience, University of Virginia, Charlottesville, VA 22908, USA 3Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 4Animal Resources Center and the Veterinary Pathology Core, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.immuni.2015.03.006 2Center

SUMMARY

Missense mutations in the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing family of gene 12 (Nlrp12) are associated with periodic fever syndromes and atopic dermatitis in humans. Here, we have demonstrated a crucial role for NLRP12 in negatively regulating pathogenic T cell responses. Nlrp12 / mice responded to antigen immunization with hyperinflammatory T cell responses. Furthermore, transfer of CD4+CD45RBhi Nlrp12 / T cells into immunodeficient mice led to more severe colitis and atopic dermatitis. NLRP12 deficiency did not, however, cause exacerbated ascending paralysis during experimental autoimmune encephalomyelitis (EAE); instead, Nlrp12 / mice developed atypical neuroinflammatory symptoms that were characterized by ataxia and loss of balance. Enhanced T-cell-mediated interleukin-4 (IL-4) production promotes the development of atypical EAE disease in Nlrp12 / mice. These results define an unexpected role for NLRP12 as an intrinsic negative regulator of T-cell-mediated immunity and identify altered NF-kB regulation and IL-4 production as key mediators of NLRP12-associated disease. INTRODUCTION Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) compose a family of intracellular sensor molecules that are involved in the regulation of inflammatory signaling in response to infection and cellular stress (Kanneganti, 2010; Kanneganti et al., 2007; Lukens et al., 2012). Recent studies have revealed pivotal roles for NLR-mediated inflammation in a spectrum of diverse human autoimmune and inflammatory disorders (Kanneganti and Dixit, 2012; Lamkanfi et al., 2011; Lukens et al., 2011). The vast majority of NLRs to date have been defined as activators of inflammatory signaling in innate immune cells. For instance, the prototypical NLR members NOD1 and NOD2 654 Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc.

initiate proinflammatory NF-kB and mitogen activated protein kinase (MAPK) signaling in response to their direct recognition of bacterial peptidoglycan fragments (Strober et al., 2006). Multiple NLRs have also been described to promote the activation and secretion of interleukin-1b (IL-1b) and IL-18 by coordinating the assembly of the inflammasome complex (Shaw et al., 2011; Strowig et al., 2012). Negative regulation of inflammatory signaling has also recently been ascribed to NLRP6, NLRP12, and NLRX1 (Anand et al., 2012; Chen et al., 2011; Elinav et al., 2011; Lei et al., 2012; Normand et al., 2011; Xia et al., 2011; Zaki et al., 2011). However, the cellular and molecular mechanisms that direct the suppression of inflammation by this new class of inhibitory NLRs remain to be formally elucidated. NLRP12 is a recently identified member of this class of inhibitory NLR proteins. Missense mutations in Nlrp12 lead to periodic fever syndromes and atopic dermatitis in humans (Borghini et al., 2011; Je´ru et al., 2008, 2011; Macaluso et al., 2007), although little is known about how defective NLRP12-mediated signaling contributes to these autoinflammatory disorders. In vitro studies with human and mouse macrophages suggest that NLRP12 is a negative regulator of toll-like receptor (TLR)-induced cytokine production (Lich et al., 2007; Williams et al., 2005; Zaki et al., 2011, 2014). Moreover, NLRP12-mediated suppression of proinflammatory signaling was recently shown to play a central role in the attenuation of colon inflammation and tumorigenesis in mice (Allen et al., 2012; Zaki et al., 2011). These studies establish NLRP12 as a critical regulator of inflammatory responses in innate immune cells. However, the putative role and importance of NLRP12 in regulating T cell responses during inflammatory disease progression has not been characterized. T cells are centrally involved in the pathogenesis of numerous autoinflammatory diseases, to which they participate in both tissue destruction and the sustained recruitment of inflammatory cells through their release of effector cytokines and chemokines (Goverman, 2009). In this study, we focused on investigating the pathophysiological role of NLRP12 in shaping autoinflammatory T cell responses. Deficiency in NLRP12 promoted the generation of hyperinflammatory T cells in response to antigen immunization. Nlrp12 / mice did not, however, exhibit exacerbated demyelinating disease in the T-cell-driven experimental autoimmune encephalomyelitis (EAE) mouse model. Rather, the absence of NLRP12 provoked an alternative form of neuroinflammation

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Figure 1. NLRP12 Deficiency Promotes the Generation of Hyperinflammatory T Cell Responses WT and Nlrp12 / mice were immunized with MOG peptide in CFA adjuvant and mice were harvested on day 27. (A) Splenocytes were restimulated directly ex vivo and the intracellular production of IFN-g, IL-17, and TNF-a by CD4+ T cells was determined. Pooled data are presented in the right panel. (B) Splenocytes were stimulated for 48 hr with MOG peptide and cytokine production was measured by ELISA. (C) Expression of activation markers by splenic CD4+ T cells. Data are representative of at least four independent experiments with three to six mice per group. All graphs depict mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; Student’s t test. See also Figure S1.

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that was characterized by atypical EAE symptoms that included loss of balance and ataxia. This development of atypical demyelinating neuroinflammation in Nlrp12 / mice was found to be dependent on dysregulated IL-4 production and we further identify NLRP12 as an intrinsic negative regulator of T cells. RESULTS NLRP12 Negatively Regulates In Vivo T Cell Responses NLRs have emerged as central regulators of inflammatory signaling, and multiple NLRs have been identified as critically involved in the regulation of proinflammatory cytokine production by antigen-presenting cells. In contrast, the ability of NLRs to modulate T cell responses and how NLR-dependent control of T cells influences autoinflammatory disease progression remains poorly defined. Thus, to elucidate the role of NLRP12 in shaping T cell responses, wild-type (WT) and NLRP12-deficient mice were immunized with MOG peptide in CFA adjuvant. T cells isolated from the spleens of Nlrp12 / mice at day 27 after immunization displayed a hyperinflammatory phenotype and produced higher amounts of interferon-g (IFN-g), IL-17, and tumor necrosis factor-a (TNF-a) after restimulation (Figure 1A). Likewise, Nlrp12 / splenocytes were also found to secrete increased amounts of cytokines after stimulation with MOG peptide (Figure 1B). Surface marker expression analysis revealed that Nlrp12 / T cells expressed higher amounts of CD44 and CD69, and markedly downregulated CD62L relative to WT cells (Figure 1C), which suggests that T cells are in a hyperactivated state in NLRP12-deficient mice. To further characterize the abil-

ity of NLRP12 to modulate T cell responses in a separate model, we evaluated T cell activation status and effector cytokine production after DSS-induced inflammation. In agreement with what was observed in the antigen immunization model, Nlrp12 / T cells isolated from dextran sodium sulfate (DSS)-challenged mice also exhibited a more activated phenotype (CD44hiCD62Llo) and produced greater amounts of inflammatory cytokines (Figure S1). Collectively, these results establish NLRP12 as a negative regulator of T cell activation in models of antigen immunization and DSS-induced colitis. NLRP12-Deficient Mice Develop Atypical EAE Disease To delineate the role of NLRP12 in the regulation of T-cell-driven autoimmune disease progression, we evaluated demyelinating neuroinflammatory disease in the experimental autoimmune encephalomyelitis (EAE) model. Given our initial observation that Nlrp12 / T cells were hyperinflammatory after antigen immunization (Figure 1), we hypothesized that NLRP12-deficient mice would develop exacerbated neuroinflammatory disease. In contrast to our expectations, classical clinical disease scores that are based on ascending paralysis were consistently lower in Nlrp12 / mice (Figure 2A). Instead, Nlrp12 / mice presented with atypical symptoms of neuroinflammatory disease that included loss of balance and ataxia (Figure 2B and Movies S1 and S2). Histological analysis revealed that atypical neuroinflammatory disease in Nlrp12 / mice was associated with enhanced mononuclear cuffing in the brain (Figures 2C and 2D). Likewise, protection against paralyzing demyelination in NLRP12-deficient mice was further confirmed by Luxol fast blue staining (LFB) of spinal cord sections (Figure S2). Consistent with previous studies that linked atypical EAE disease with extensive infiltration of neutrophils into the central nervous system (CNS) (Kroenke et al., 2010; Stromnes et al., 2008), neutrophils also represented an increased fraction of the infiltrating myeloid cell population in Nlrp12 / mice (Figures 2E and 2F). Furthermore, Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc. 655

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the proportions of neutrophils to other myeloid cells were increased in the periphery of diseased Nlrp12 / mice (Figure 2G). In addition, non-classical neuroinflammatory disease in Nlrp12 / mice was associated with increased frequencies of Foxp3-expressing regulatory T (Treg) cells in both the periphery (Figure 2H) and the CNS (Figure 2I). Although multiple NLRs have been identified to play important roles in the orchestration of inflammasome-mediated cytokine production (Vladimer et al., 2012), atypical EAE disease in Nlrp12 / mice was not associated with altered generation of IL-1b or IL-18 (Figure S3). Dysregulated IL-4 Production Promotes Atypical Neuroinflammatory Disease in Nlrp12–/– Mice To investigate the immunological factors that contribute to the development of non-classical neuroinflammatory disease symptoms in Nlrp12 / mice, we next explored whether differences in clinical disease parameters were associated with differential regulation of T-cell-mediated cytokine production in the CNS. Deficiency in NLRP12 was not, however, found to affect the production of IFN-g, IL-17, or TNF-a by CD4+ T cells in the CNS (Fig656 Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc.

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WT and Nlrp12 / mice were immunized with MOG+CFA and pertussis toxin to induce EAE. (A) Classical clinical scores that are based on ascending paralysis. (B) Percentages of mice that developed classical (ascending paralysis) or atypical (ataxia and impaired balance) EAE disease symptoms. (C) H&E staining of brain lesions. The arrows highlight areas of extensive mononuclear cuffing (original magnification 3 20). (D) Mononuclear cuffing histology scores from WT (n = 7) and Nlrp12 / (n = 5) brain sections. (E) Frequencies of CNS-infiltrating cells that are neutrophils (CD11b+Gr-1+) or other myeloid cells (CD11b+Gr-1 ) on day 28. (F) Ratios of neutrophils to other myeloid cells in the CNS. (G) Ratios of neutrophils to other myeloid cells in the spleen. (H and I) Frequencies of CD4+ T cells that express Foxp3 in the spleen (H) and CNS (I). Each point represents an individual mouse. Data are representative of four independent experiments with three to six mice per group. Statistical significance in (A) was determined by two-way ANOVA. Statistical significance in (D) and (F)–(I) was determined by Student’s t test. All graphs depict mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S2 and Movies S1 and S2.

_______ * ure 3A). Our knowledge of the cellular and molecular mediators that potentiate atypical EAE currently remains limited, but a few studies have pointed to important roles for IL-4 in the induction of atypical neuroinflammation (Delgoffe et al., 2011; Nlrp12-/WT Wensky et al., 2001). Therefore, we next assessed whether dysregulated IL-4 production promoted atypical neuroinflammatory disease in Nlrp12 / mice. We observed greater production of IL-4 by both CNS and splenic CD4+ T cells in Nlrp12 / mice (Figures 3A and 3B). Furthermore, restimulation of NLRP12-deficient splenocytes with myelin-specific antigen provoked markedly enhanced secretion of IL-4 (Figure 3C). To confirm that dysregulated IL-4 production is indeed involved in dampening ascending paralysis while conversely spurring the development of atypical EAE disease symptoms in Nlrp12 / mice, we treated mice with anti-IL-4 neutralizing antibody to disrupt IL-4-mediated signaling. In agreement with published reports (Bettelli et al., 1998; Falcone et al., 1998; Furlan et al., 1998; Shaw et al., 1997), blockade of IL-4 during EAE caused exacerbated classical disease symptoms in WT mice (Figure 3D). Furthermore, ablation of IL-4 during EAE caused NLRP12-deficient mice to develop classical symptoms of demyelinating neuroinflammatory disease that included severe ascending paralysis and morbidity (Figure 3D). Collectively these results suggest a causal role for hyperinflammatory IL-4 production in driving atypical EAE disease progression in Nlrp12 / mice.

Figure 3. Dysregulated IL-4 Production Promotes Atypical Neuroinflammatory Disease in Nlrp12–/– Mice

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NLRP12 Is Dispensable for Thymic Development To elucidate the level at which NLRP12 influences T cell activation, we first evaluated the influence of NLRP12 deficiency on T cell development. NLRP12 deficiency was not found to promote major perturbations in positive T cell selection under homeostatic conditions, and standard thymic development flow cytometry gating strategies revealed normal distribution of thymocytes in Nlrp12 / mice (Figures S4A and S4B). Moreover, genetic deletion of Nlrp12 failed to alter the accumulation of CD4 CD8 , CD4+CD8+, CD4+CD8 , or CD4 CD8+ thymocytes (Figure S4C). Analysis of the double-negative (DN) population via CD44 and CD25 discrimination also ruled out a homeostatic role for NLRP12 in negative selection (Figures S4D and S4E). These results indicate that NLRP12 is dispensable for normal thymic development and suggest a role for NLRP12 in the regulation of mature T cell responses. NLRP12 Shapes Peripheral T Cell Development in Naive Mice To formally address whether the absence of NLRP12 causes alterations in peripheral T cell development and activation under homeostatic conditions, we next evaluated T cell numbers, cytokine production, and Treg cell frequencies in naive mice. We detected enhanced numbers of peripheral CD4+ and CD8+ T cells in Nlrp12 / mice (Figure S4F). NLRP12-deficient T cells also displayed a more inflammatory phenotype and produced greater amounts of IFN-g and IL-17 after brief restimulation (Figure S4G). Consistent with a role for NLRP12 in shaping T cell development in the periphery, we also observed greater frequencies of Foxp3-expressing Treg cells in naive Nlrp12 / mice (Figure S4H). These results indicate that NLRP12 is involved in the regulation of mature T cell development and activation. However, NLRP12 appeared to exert a more prominent effect on T cells responses after overt stimula-

WT and Nlrp12 / mice were immunized with MOG+CFA and pertussis toxin to induce EAE and mice were harvested either on day 27 (A and B) or day 29 (C). (A) Frequencies of CD4+ T cells that express IFN-g, IL-17, TNF-a, or IL-4 in the CNS. (B) Frequencies of IL-4-producing CD4+ T cells in the spleen. (C) Splenocytes were stimulated with MOG peptide for 48 hr and IL-4 production was measured by ELISA. (A–C) Data are representative of three independent experiments with three to six mice per group. Statistical significance was determined by Student’s t test. (D) Classical ascending paralysis clinical scores for WT and Nlrp12 / EAE mice that were treated with either vehicle control or 500 mg of anti-IL-4 blocking antibody on days 1, 0, 2, 4, 6, 8, and 13. Data are representative of two independent experiments with 5–14 mice per group. Statistical significance was determined by two-way ANOVA. All graphs depict mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S3.

tion, as was observed in the antigen immunization model (Figure 1). NLRP12 Is an Intrinsic Negative Regulator of T Cell Activation We were next interested in determining whether NLRP12 modulates T cell activation through an extrinsic role in antigen-presenting cells (APCs) or through an intrinsic function in T cells. As a first approach we evaluated the expression of Nlrp12 mRNA transcripts in various immune cell populations in an effort to help identify the cell types that NLRP12 could potentially function in to shape T cell responses. We found that Nlrp12 mRNA was most highly expressed in polymorphonuclear leukocytes (PMNs), CD4+ T cells, and CD8+ T cells (Figure 4A). Nlrp12 mRNA expression was also detected in resting dendritic cells and macrophages, but its expression in these antigen-presenting cells was markedly less in comparison to T cells and PMNs. To ascertain whether the observed expression of Nlrp12 in T cells influences functional responses, in vitro T cell thymidine incorporation assays were conducted. Purified Nlrp12 / T cells displayed more extensive thymidine incorporation than WT T cells in response to anti-CD3 stimulation (Figure 4B), which implicates NLRP12 in the attenuation of T cell expansion. It is possible that defects in Treg-cell-mediated suppressive functions are responsible for the hyperactive T cell responses that are observed in Nlrp12 / mice. Therefore, to formally test this possibility, in vitro Treg cell suppression assays were conducted. In agreement with our anti-CD3 thymidine incorporation results, we also observed enhanced proliferation by CFSE dye dilution of Nlrp12 / T cells in the absence of Treg cells. Furthermore, the suppressive capacity exhibited by Treg cells that lacked NLRP12 was equal to that of WT Treg cells in classical in vitro suppression assays (Figure 4C), and Nlrp12 / Treg cells Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc. 657

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(A) Relative expression of Nlrp12 in purified macrophages (Ly6G CD11b+), dendritic cells (CD11c+MHCIIhi), CD4+ T cells (Thy1.2+CD4+), CD8+ T cells (Thy1.2+CD8+), and polymorphonuclear leukocytes (PMNs) (Ly6G+CD11b+). (B) Purified naive T cells isolated from WT and Nlrp12 / mice were stimulated with plate bound anti-CD3 for 48 hr and the incorporation of thymidine was measured during the final 8 hr. (C) Purified conventional CD4+ T cells (CD4+ CD62Lhi) and Treg cells (CD4+CD25+) were isolated from WT and Nlrp12 / mice by flow cytometry sorting. Conventional CD4+ T cells were labeled with CFSE and stimulated with anti-CD3 and irradiated splenocytes. Treg cell suppression assays were then conducted by mixing conventional CD4+ T cells and Treg cells in various combinations and at different ratios. All graphs depict mean ± SEM. *p < 0.05, **p < 0.01; Student’s t test. See also Figures S4 and S5.

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did not display any impairment in IL-10 production (Figure S4I). Conversely, NLRP12-deficient T cells were less responsive to the suppression exerted by increasing concentrations of WT Treg cells, and conventional Nlrp12 / T cells were more likely to undergo division even when WT Treg cells were present at high numbers (Figure 4C). These findings indicate that the altered hyperinflammatory T cell responses that were observed in Nlrp12 / mice are most likely not the result of dysfunctional Nlrp12 / Treg cell responses. Rather, they indicate that dysregulated Nlrp12 / effector T cells are responsible for the observed T-cell-mediated effects in NLRP12-deficient mice. NLRP12 Is an Intrinsic Negative Regulator of In Vivo T Cell Responses To confirm an intrinsic role for NLRP12 in the regulation of T cell responses in vivo, 1:1 competitive adoptive T cell transfer assays were carried out. For these experiments, congenically mismatched WT and NLRP12-deficient T cells were adoptively transferred into Rag1 / recipient mice. After 2 weeks of homeostatic expansion, CD45.2+ Nlrp12 / T cells markedly outcompeted CD45.1+ WT T cells in both the lymph nodes (LNs) and spleens of recipient mice (Figure S5A). Nlrp12 / T cells also expressed greater amounts of proinflammatory cytokines on a per-cell basis (Figure S5B). To ascertain whether NLRP12 also 658 Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc.

intrinsically controls T cell responses under pathophysiological conditions, we generated 1:1 chimera mice with Nlrp12-/- CD4+ T cells CD45.2+ Nlrp12 / and CD45.1+ WT WT Treg cells bone marrow cells. After reconstitution, mice were immunized with MOG peptide plus CFA adjuvant and T cell responses were monitored in the spleen at day 21. Nlrp12-/- CD4+ T cells Nlrp12-/- Treg cells Intriguingly, NLRP12-deficient T cells outcompeted WT T cells in the periphery after stimulation (Figure S5C). Furthermore, T cells produced greater Nlrp12 / amounts of IFN-g and IL-17 on a percell basis in the 1:1 chimeric mice (Figure S5C). These results establish an unexpected, intrinsic role for NLRP12 in the negative regulation of T cell responses under pathophysiological conditions. NLRP12 Can Regulate Inflammatory Disease through T-Cell-Intrinsic Functions Defective NLRP12 expression was shown to promote exacerbated inflammatory disease in models of colon damage and tumorigenesis (Zaki et al., 2011). However, the cellular mechanisms responsible for enhanced inflammation in Nlrp12 / mice have not been formally elucidated. To ascertain whether Nlrp12 / T cells directly promote hyperinflammation and disease pathology, we transferred CD4+CD45RBhi T cells from WT or Nlrp12 / mice into Rag1 / mice and tracked the development of colitis. In comparison to the injection of WT T cells, adoptive transfer of Nlrp12 / T cells provoked more severe weight loss (Figure 5A) and intestinal inflammation (Figures 5B and 5C). The development of aggravated disease in Nlrp12 / T cell transfer mice was associated with enhanced recruitment of inflammatory cells and cell death in the afflicted mucosa (Figures 5D and S6). Adoptive transfer of Nlrp12 / CD4+CD45RBhi T cells into Rag1 / mice also provoked the development of atopic dermatitis in recipient mice (Figure 5E), and this was

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Purified CD4+CD45RBhi WT or Nlrp12 / T cells were adoptively transferred into Rag1 / mice. (A) Percent weight change over time. (B) Representative large intestine histology at 9 weeks after adoptive transfer of CD4+CD45RBhi T cells (original magnification 3 20). (C) Combined colon histology scores. (D) Representative immunohistochemistry staining of T cells (anti-CD3), macrophages (anti-F4/80), neutrophils (anti-Gr-1), and cell death (TUNEL) in inflamed colon tissue (original magnification 3 20). (E) Representative H&E ear sections (original magnification 3 20). (F) Combined ear histology scores. Each point represents an individual mouse and the line represents the mean ± SEM. Data are representative of four independent experiments with three to seven mice per genotype. All graphs depict mean ± SEM. *p < 0.05, **p < 0.01; Student’s t test. See also Figure S6.

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associated with more severe skin pathology (Figure 5F). Cutaneous inflammation in this model is typically associated with dysregulated T helper 2 (Th2) cell-associated inflammation (Davenport et al., 2002; Leon et al., 2006; Scho¨n et al., 1997), suggesting that altered IL-4 production might also be contributing to disease pathology. We next determined the mechanisms by which Nlrp12 / T cells induce intestinal inflammation and atopic dermatitis. We found that Nlrp12 / T cells accumulated in greater numbers (Figure 6A) and exhibited a more activated phenotype (Figure 6B) in the T cell transfer colitis model. To investigate the contributions of altered inflammatory cytokine production in exacerbated disease progression, we evaluated cytokine production after overnight (16 hr) anti-CD3 stimulation. T cell receptor (TCR)mediated activation of cells isolated from Nlrp12 / CD4+ CD45RBhi T cell transfer mice resulted in more robust secretion of proinflammatory cytokines including IL-6, TNF-a, and GMCSF (Figure 6C, left). Furthermore, Nlrp12 / T cells produced

potent amounts of Th2-cell-associated cytokines including IL-4, IL-5, and IL-13 (Figure 6C, right), which helps to explain the development of atopic dermatitis in these mice. Consistent with the more pronounced production of Th2-cell-derived cytokines in Nlrp12 / T cell transfer mice, the expression of YM1 and MBP, which are markers that have been associated with Th2-cell-mediated inflammation, were also markedly increased in the CD4+CD45RBhi colons of Nlrp12 / T cell transfer mice (Figure S7; Ponomarev et al., 2007). These results identify NLRP12 as a negative regulator of T-cell-induced inflammatory disease and suggest that unchecked T-cell-mediated cytokine production centrally contributes to disease pathology. Because our findings in the CD4+CD45RBhi T cell transfer model of colitis indicated that NLRP12 can influence autoinflammatory disease pathogenesis through specific actions in T cells, we were next interested in elucidating whether NLRP12-dependent regulation of T cells responses contributed to the altered EAE disease phenotype that was observed in Nlrp12 / mice. To this end we evaluated the development of demyelinating neuroinflammatory disease in the passive transfer model of EAE disease. Consistent with our previous data showing abrogated classical EAE disease in germline NLRP12-deficient mice (Figure 2), the transfer of enriched myelin-specific Nlrp12 / T cells into Rag1 / recipient mice resulted in less severe paralyzing neuroinflammatory disease (Figure 6D). As an independent approach, WT or Nlrp12 / thymocytes were also adoptively transferred into T-cell-deficient mice (Tcra / mice) and EAE was induced. In agreement with our previous results, Tcra / mice that were reconstituted with Nlrp12 / thymocytes were also protected from severe paralyzing EAE disease Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc. 659

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Figure 6. Dysregulated T Cell Activation and Cytokine Production Contribute to the Pathogenicity of Nlrp12–/– T Cells Purified CD4+CD45RBhi WT or Nlrp12 / T cells were adoptively transferred into Rag1 / mice. (A) Total numbers (mean ± SEM) of CD4+ T cells in the mesenteric LNs (MLNs) and spleen at 9 weeks after adoptive transfer. (B) Expression of CD69 by adoptively transferred CD4+ T cells in the MLNs and spleen. (C) WT (n = 5) and Nlrp12 / (n = 5) splenocytes were stimulated overnight with anti-CD3 and cytokine production was measured by ELISA. (D) Passive transfer EAE was induced in Rag1 / recipient mice by transferring enriched myelinspecific CD4+ T cells that were generated and expanded from WT and Nlrp12 / mice. Classical signs of EAE disease (ascending paralysis) were assessed over time. (E) T-cell-deficient (Tcra / ) mice were reconstituted with WT or Nlrp12 / thymocytes. Mice were then immunized with MOG plus CFA to induce EAE, and the development of classical signs of clinical disease (ascending paralysis) were assessed over time. All graphs depict mean ± SEM. *p < 0.05, **p < 0.01; Student’s t test. See also Figure S7.

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(Figure 6E). These studies in T cell transfer models of disease thus implicate critical T-cell-intrinsic roles for NLRP12 in the regulation of autoinflammation and autoimmune disease pathogenesis. NLRP12 Negatively Regulates NF-kB Signaling in T Cells Previous studies suggest that NLRP12 dampens TLR-induced NF-kB activation in macrophages and dendritic cells (Lich et al., 2007; Williams et al., 2005; Zaki et al., 2011), but the specific molecular pathways that are controlled by NLRP12 in T cells remain unknown. Many of the cytokines that were found to be enhanced in Nlrp12 / T cell transfer colitis mice are known to be regulated by NF-kB-dependent signaling (Figure 6C), so we were interested in determining whether NLRP12 limits T-cell-induced inflammatory disease through its regulation of NF-kB activation. Indeed, transfer of Nlrp12 / CD4+CD45RBhi T cells resulted in enhanced phosphorylation of IkBa (Figures 7A and 7B). Moreover, we also detected greater NF-kB2 processing (conversion of p100 to p52) in Nlrp12 / CD4+CD45RBhi T cell transfer colon lysates, which indicates 660 Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc.

enhanced non-canonical NF-kB signaling in the absence of NLRP12 (Figures 7A and 7B). To elucidate whether NLRP12 is involved in the global dampening of proinflammatory signaling or whether it is specifically involved in the NF-kB pathway, we also evaluated the regulation of other important signaling pathways that are known to drive disease pathology in the T cell transfer colitis model. Exacerbated inflammatory disease in CD4+CD45RBhi Nlrp12 / T cell transfer mice was not, however, associated with altered ERK, p38, or STAT3 signaling (Figures 7A and 7B). Importantly, we also observed enhanced canonical and non-canonical NF-kB signaling in the brains of NLRP12-deficient mice during EAE (Figures 7C and 7D). To determine whether NLRP12-mediated regulation of NF-kB activation in T cells potentially contributes to the altered inflammatory signaling that was observed in these two models of autoimmunity, we evaluated signaling in purified T cells. We found that stimulation of Nlrp12 / T cells with anti-CD3 plus antiCD28 led to enhanced phosphorylation of IkBa and greater amounts of processing of p100 to p52 (Figure 7E). Furthermore, pharmacological inhibition of NF-kB activation with SC514 treatment rescued the hyperproliferation exhibited by Nlrp12 / T cells stimulated with anti-CD3 plus anti-CD28 (Figure 7F). Inhibition of NF-kB signaling with SC514 was also found to return IL-4 secretion by Nlrp12 / T cells to wild-type amounts (Figure 7G). Collectively, these findings identify NLRP12 as a negative regulator of both canonical and non-canonical NF-kB signaling in T cells.

A

C

E

Figure 7. NLRP12 Negatively Regulates Both Canonical and Non-canonical NF-kB Signaling

B

D

F

G

DISCUSSION Mutations in Nlrp12 have been described to cause autoinflammatory disorders in humans (Borghini et al., 2011; Je´ru et al., 2008, 2011; Macaluso et al., 2007), but the cellular and molecular mechanisms involved in NLRP12-mediated regulation of immune responses remain poorly defined. In this study, we focused on elucidating the roles of NLRP12 in shaping inflammatory T cell responses and T-cell-mediated disease. We discovered that the induction of experimental autoimmune encephalomyelitis (EAE) in NLRP12-deficient mice provoked the generation of hyperinflammatory myelin-specific T cell responses. Nlrp12 / T cells did not, however, cause exacerbated demyelinating disease in the T-cell-driven EAE model. In contrast, Nlrp12 / mice developed atypical neuroinflammatory disease symptoms that included loss of balance and ataxia. Moreover, the development of non-classical EAE disease in mice that lack NLRP12 was characterized by altered brain

(A and B) Purified CD4+CD45RBhi WT or Nlrp12 / T cells were adoptively transferred into Rag1 / mice. Colons were collected from WT and Nlrp12 / CD4+CD45RBhi transfer mice at 9 weeks after adoptive transfer. (A) Immunoblot analysis of colon lysates. Each lane represents an individual mouse. (B) Densitometry quantification of band intensity. Data are representative of four independent experiments with three to seven mice per genotype. (C and D) WT and Nlrp12 / mice were immunized with MOG+CFA and pertussis toxin to induce EAE and mice were harvested on day 29. Immunoblot analysis of brain lysates. (D) Densitometry quantification of band intensity. Data are representative of three independent experiments with three to five mice per genotype. (E–G) Purified WT and Nlrp12 / T cells were stimulated with anti-CD3 (1 mg/ml) plus anti-CD28 (1 mg/ml) in the presence or absence of the NF-kB inhibitor SC514 (3 mg/ml). (E) Lysates were probed for total and phosphorylated IkBa, ERK p100/p52, and GAPDH. (F) Counts per minute (CPM) of T cells at 72 hr after stimulation with anti-CD3 and anti-CD28. (G) Secretion of IL-4 at 72 hr after stimulation with anti-CD3 and anti-CD28. All graphs depict mean ± SEM. *p < 0.05, **p < 0.01; Student’s t test.

pathology and a myeloid cell CNS infiltrate that was dominated by neutrophils. We demonstrated that NLRP12 is an intrinsic negative regulator of T cells and that NLRP12 deficiency in T cells was sufficient to induce colitis and atopic dermatitis in the CD4+CD45RBhi T cell transfer model. Mechanistically, dysregulated NF-kB activation and enhanced IL-4 production were found to contribute to T-cell-induced inflammation in Nlrp12 / mice. Our findings define a previously unknown role for NLRs in the negative regulation of T cell activation and unravel a T-cell-intrinsic function for NLRP12 in the suppression of inflammatory cytokine production. Furthermore, our discovery that NLRP12 deficiency leads to unchecked IL-4 production helps to explain how missense mutations in Nlrp12 contribute to the development of atopic dermatitis in humans. Utilization of the EAE model system has helped to define the inflammatory factors that contribute to demyelination and paralysis in multiple sclerosis (MS) pathogenesis. However, considerably less is known about the conditions and pathways that induce many of the other disease symptoms that are common in MS patients, including ataxia, impaired balance, and loss of sensation. Our findings presented here suggest that investigation of EAE in Nlrp12 / mice could provide a muchneeded model system to formally characterize the immune factors that trigger non-paralyzing neuroinflammatory disease Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc. 661

symptoms in MS patients. Importantly, excessive production of both classical inflammatory factors (IFN-g, IL-6, GM-CSF) and Th2-cell-associated cytokines (IL-4 and IL-5) have been observed in patients with severe MS (Hedegaard et al., 2008; Hohnoki et al., 1998; Link et al., 1994). However, the physiological role of Th2 cell cytokines in MS disease pathogenesis has remained elusive. In the EAE mouse model, IL-4 has been described to provide protection against classical ascending paralysis (Bettelli et al., 1998; Falcone et al., 1998; Furlan et al., 1998; Shaw et al., 1997). In agreement with these findings, we observed that enhanced IL-4 production limited the development of paralysis in NLRP12-deficient mice. Unchecked IL-4 production during EAE has also been shown to induce atypical neuroinflammatory disease symptoms (ataxia and issues with balance), which is consistent with the IL-4associated atypical neuroinflammatory disease phenotype that was observed in Nlrp12 / mice (Delgoffe et al., 2011; Wensky et al., 2001). Th2-cell-associated cytokines also play pathogenic roles in intestinal inflammatory disease. In particular, Th2 cell cytokines are highly expressed in combination with Th1-cell-associated cytokines in ulcerative colitis (UC) patients (Fuss et al., 1996), and Th2 cell cytokines are believed to be the major drivers of pathology in UC (Cho, 2008). Likewise, Th2 cell cytokines potentiate UC-like intestinal disease in the oxazolone colitis mouse model (Boirivant et al., 1998; Heller et al., 2002; Mizoguchi et al., 1999). In these studies, it was shown that Th2 cell cytokines cause inflammation to the intestinal epithelial layer and that neutralization of IL-4 with blocking antibody treatment rescues oxazolone-mediated colitis. Similarly, hyperinflammatory IL-4 production has also been described to cause exacerbated disease in the CD4+CD45RBhi T cell transfer colitis model (Fort et al., 2001). In this setting, Th2 cell cytokines promote the upregulation of major hisotocmpatibility complex (MHC) class II expression in the lamina propria of the colon and also stimulate the expression of exorbitant amounts of both Th1- and Th2-cell-associated cytokines in the intestinal mucosa. Our findings demonstrate prominent roles for NLRP12 in the attenuation of colitogenic T cell responses and identify NLRP12 as an attractive candidate for the treatment of Th2-cell-associated intestinal inflammatory disease. In summary, we identify NLRP12 as a negative regulator of inflammatory T cell responses and T-cell-mediated disease. NLRP12 intrinsically regulates T cell activation, and dysregulated NLRP12-mediated signaling in T cells is sufficient to induce inflammatory disease. However, deficiency in NLRP12 did not provoke more severe demyelinating disease and paralysis in the EAE model. In contrast, Nlrp12 / mice developed atypical neuroinflammatory disease that was characterized by worsened brain pathology and excessive production of both traditional inflammatory cytokines (IL-6, GM-CSF, TNF-a) and IL-4. Furthermore, dysregulated T-cell-mediated NF-kB signaling and downstream IL-4 were found to contribute to inflammatory responses in Nlrp12 / mice. These findings define additional roles for NLRP12 as an intrinsic attenuator of T cell responses and IL-4-associated inflammation. Moreover, they suggest that NLRP12-targeted therapies might provide a novel treatment strategy in T-cell-mediated disease. 662 Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc.

EXPERIMENTAL PROCEDURES Mice Nlrp12 / mice have been previously described (Zaki et al., 2011). Tcra / mice were kindly provided by Dr. Maureen McGargill (St. Jude Children’s Research Hospital) and Rag1 / mice were received from Dr. Terrence L. Geiger (St. Jude Children’s Research Hospital). Mice at 6–12 weeks of age were used. All mice were kept in specific-pathogen-free conditions within the Animal Resource Center at St. Jude Children’s Research Hospital. Animal protocols were approved by the Institutional Animal Care and Use Committee of St. Jude Children’s Research Hospital. Flow Cytometry Protocols and Antibodies For the flow cytometry cellular analysis, the following monoclonal antibodies were used: CD4 (L3T4), IFN-g (XMG1.2), IL-17A (eBio17B7), IL-4 (11B11), MHCII (M5/114.15.2), CD11b (M1/70), CD19 (6D5), CD44 (IM7), Ly-6G (1A8), CD25 (3C7), B220 (RA3-6B2), and Gr-1 (RB6-8C5) from eBioscience and TCR-b (H57-597), CD8 (53-6.7), Foxp3 (FJK-16 s), CD62L (MEL-14), CD69 (H1.2F3), TNF-a (MP6-XT22), CD11c (N418), CD45.1 (A20), and CD45.2 (104) from Biolegend. Nlrp12 Expression Macrophages (Ly6G CD11b+), dendritic cells (CD11c+MHCIIhi), CD4+ T cells (Thy1.2+CD4+), CD8+ T cells (Thy1.2+CD8+), and polymorphonuclear leukocytes (PMNs) (Ly6G+CD11b+) were purified by flow cytometry sorting and total RNA was isolated with Trizol (Invitrogen) according to the manufacturer’s instructions. 1 mg of RNA was reverse transcribed to cDNA with random RNAspecific primers using the high-capacity cDNA reverse transcription kit (Applied Biosystems). Transcript amounts of Nlrp12 and Gapdh were analyzed with SYBR-Green (Applied Biosystems) according to the manufacturer’s recommendations. Thymidine Incorporation Assay Thy1.2+ T cells were isolated from WT and Nlrp12 / mice via positive magnetic bead enrichment (Miltenyi Biotec). Naive T cells (CD44loCD62Lhi) were then purified via FACS sorting. Purified naive T cells were stimulated with increasing concentrations of plate bound anti-CD3 in triplicate wells for 48 hr. During the last 8 hr of stimulation, T cells were pulsed with [3H]thymidine and the amount of incorporated [3H]thymidine was measured as counts per minute (cpm). Mixed Bone Marrow Chimeras Bone marrow from WT (CD45.1+) and Nlrp12 / (CD45.2+) mice was flushed from the femurs, filtered through a 40 mm filter, and mixed at a 1:1 ratio. Mixed bone marrow cells (total 5–10 3 106 cells) were transferred by tail vein injection into lethally irradiated (1,000 rad) Rag1 / mice. 1:1 bone marrow chimera mice were allowed to reconstitute for 2 months before immunization with MOG peptide in CFA. Congenic CD45 markers were utilized to verify chimerism. CD4+CD45RBhi T Cell Transfer Colitis Model Splenocytes and peripheral LNs were harvested from WT and Nlrp12 / mice and bulk T cells were positively enriched with anti-Thy1.2 magnetic beads (Miltenyi Biotec). CD4+CD45RBhi T cells were purified from the bulk T cell population by FACs sorting. Rag1 / recipient mice received 5 3 105 WT or Nlrp12 / CD4+CD45RBhi T cells by intraperitoneal injection and mice were weighed once every week to measure percent weight change. EAE Model Mice were immunized subcutaneously with 100 mg MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA (Difco Laboratories) with 500 mg Mycobacterium tuberculosis on day 0. Mice also received 200 ng of pertussis toxin (List Biological Laboratories) by intraperitoneal injection on days 0 and 2. Classical disease severity was assessed daily by assigning clinical scores according to the following ascending paralysis scale: 0, no disease; 1, tail paralysis; 2, weakness of hind limbs; 3, paralysis of hind limbs; 4, paralysis of hind limbs and severe hunched posture; 5, moribund or death. The incidence of atypical disease was recorded based on the development of balance

issues and ataxia. To evaluate the role of IL-4 in Nlrp12-mediated atypical neuroinflammatory disease, EAE was induced in WT and Nlrp12 / mice as described above. Mice received either vehicle control or 500 mg anti-IL-4 blocking antibody (11B11) by intraperitoneal dosage on days 1, 0, 2, 4, 6, 8, and 13, and classical clinical scores were assigned based on ascending paralysis development. Additional Supplemental Experimental Procedures, including histopathology and immunohistochemistry, competitive T cell adoptive transfer, chronic DSS model, Treg cell suppression assay, passive and thymocyte transfer EAE transfer models, and CFSE T cell proliferation assay, are available online.

Chen, G.Y., Liu, M., Wang, F., Bertin, J., and Nu´n˜ez, G. (2011). A functional role for Nlrp6 in intestinal inflammation and tumorigenesis. J. Immunol. 186, 7187– 7194.

Statistics All results are presented as mean ± SEM. Two-way ANOVA analysis was utilized to evaluate differences in clinical EAE scores over time. All other p values were calculated by two-tailed unpaired Student’s t tests. p values < 0.05 were considered significant. p values are denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

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SUPPLEMENTAL INFORMATION

Elinav, E., Strowig, T., Kau, A.L., Henao-Mejia, J., Thaiss, C.A., Booth, C.J., Peaper, D.R., Bertin, J., Eisenbarth, S.C., Gordon, J.I., and Flavell, R.A. (2011). NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell 145, 745–757.

Supplemental Information includes seven figures, Supplemental Experimental Procedures, and two movies and can be found with this article online at http:// dx.doi.org/10.1016/j.immuni.2015.03.006.

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AUTHOR CONTRIBUTIONS J.R.L., P.G., and T.-D.K. designed the study. J.R.L., P.G., P.J.S., M.H.Z., and M.J.B. performed experiments. P.V. performed and analyzed the histopathology data. S.A.B. made IL-4 neutralizing antibodies. H.C. provided essential reagents and scientific insights. J.R.L., P.G., and T.-D.K. analyzed data and wrote the manuscript. ACKNOWLEDGMENTS P.G. is a postdoctoral fellow supported by Paul Barrett Endowed Fellowship from St. Jude Children’s Research Hospital. This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, part of the NIH, under Award Number AR056296 (T.-D.K.); the National Cancer Institute, part of the NIH, under Award Number CA163507 (T.-D.K.); the National Institute of Allergy and Infectious Diseases, part of the NIH, under Award Number AI101935 (T.-D.K.); and ALSAC. Received: September 29, 2014 Revised: January 9, 2015 Accepted: March 18, 2015 Published: April 14, 2015 REFERENCES Allen, I.C., Wilson, J.E., Schneider, M., Lich, J.D., Roberts, R.A., Arthur, J.C., Woodford, R.M., Davis, B.K., Uronis, J.M., Herfarth, H.H., et al. (2012). NLRP12 suppresses colon inflammation and tumorigenesis through the negative regulation of noncanonical NF-kB signaling. Immunity 36, 742–754. Anand, P.K., Malireddi, R.K., Lukens, J.R., Vogel, P., Bertin, J., Lamkanfi, M., and Kanneganti, T.D. (2012). NLRP6 negatively regulates innate immunity and host defence against bacterial pathogens. Nature 488, 389–393. Bettelli, E., Das, M.P., Howard, E.D., Weiner, H.L., Sobel, R.A., and Kuchroo, V.K. (1998). IL-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of IL-10- and IL-4-deficient and transgenic mice. J. Immunol. 161, 3299–3306. Boirivant, M., Fuss, I.J., Chu, A., and Strober, W. (1998). Oxazolone colitis: a murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4. J. Exp. Med. 188, 1929–1939. Borghini, S., Tassi, S., Chiesa, S., Caroli, F., Carta, S., Caorsi, R., Fiore, M., Delfino, L., Lasiglie`, D., Ferraris, C., et al. (2011). Clinical presentation and pathogenesis of cold-induced autoinflammatory disease in a family with recurrence of an NLRP12 mutation. Arthritis Rheum. 63, 830–839.

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