NLRP3 Inflammasome Plays a Redundant Role with Caspase 8 to Promote IL-1Β–Mediated Osteomyelitis

NLRP3 inflammasome plays a redundant role with caspase 8 to promote IL-1β–mediated osteomyelitis

Prajwal Gurunga, Amanda Burtona, and Thirumala-Devi Kannegantia,1

aDepartment of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105

Edited by Vishva M. Dixit, Genentech, San Francisco, CA, and approved March 9, 2016 (received for review February 1, 2016) Missense mutation in the proline-serine-threonine phosphatase- for CRMO are limited to the use of nonsteroidal antiinflammatory cmo interacting protein 2 (Pstpip2) gene results in the development of drugs (NSAIDs) and bisphosphonates (14). Pstpip2 mice have spontaneous chronic bone disease characterized by bone defor- proved to be a valuable tool in understanding the molecular mity and inflammation that is reminiscent of patients with chronic mechanisms involved in instigation of CRMO and bone diseases in cmo multifocal osteomyelitis (cmo). Interestingly, this disease is specif- general. Using Pstpip2 mice as a model to understand the eti- ically mediated by IL-1β but not IL-1α. The precise molecular path- ology of bone diseases, we previously demonstrated that IL-1R cmo ways that promote pathogenic IL-1β production in Pstpip2 mice signaling completely protected the progression of disease in these remain unidentified. Furthermore, how IL-1β provokes inflamma- mice (1). Adding to these studies, we and others showed that IL-1β, cmo tory bone disease in Pstpip2 mice is not known. Here, we dem- but not IL-1α, was important for induction of bone disease in these onstrate that double deficiency of Nod like receptor family, pyrin cmo mice (1, 15). More recent studies from our laboratory elucidated domain containing 3 (NLRP3) and caspase 8 in Pstpip2 mice a rather surprising redundant role for caspase 1 and caspase 8 provides similar protection as observed in caspase-1 and caspase- β cmo in the processing of IL-1 (2). The redundant roles of caspase 8–deficient Pstpip2 mice, demonstrating redundant roles for 1 and caspase 8 suggest that these caspases are functioning the NLRP3 inflammasome and caspase 8 in provoking osteomy- cmo independently in separate complexes to process the pathogenic elitic disease in Pstpip2 mice. Consistently, immunofluorescence cytokine IL-1β, unlike several other studies where caspase 1 studies exhibited distinct caspase-1 and caspase-8 puncta in dis- and caspase 8 have been proposed to be in the same complex Ptpn6spin eased neutrophils. Data from our chimera studies demon- (16). In this study, we sought to understand the upstream β strated that IL-1 produced by hematopoietic cells is sensed by the molecules that assemble the caspase-1 and caspase-8 complexes. radioresistant compartment to promote bone disease. Furthermore, Furthermore, we undertook an innovative chimeric approach to our results showed that the IL-1β signaling is unidirectional and β elucidate the IL-1R signaling feedback loop in hematopoietic and feedback signaling of IL-1 onto the hematopoietic compartment radioresistant compartments and determine how IL-1β provoked cmo is not important for disease induction. In conclusion, our studies inflammatory osteomyelitis in Pstpip2 mice. have uncovered the combined actions of the NLRP3 inflammasome We demonstrated that the caspase-1 complex is assembled by and caspase 8 leading to IL-1β maturation and the directionality of cmo NLRP3, whereas caspase 8 does not engage the TNF signaling IL-1β in driving disease in Pstpip2 mice. pathways, suggesting involvement of an alternate complex. Importantly, the NLRP3 inflammasome and caspase 8 play NLRP3 | inflammasome | caspase 8 | caspase 1 | PSTPIP2 redundant roles in IL-1β processing and inducing disease in cmo Pstpip2 mice. Activation of distinct caspase-1 and -8 com- utoinflammatory bone diseases that include osteoporosis, plexes within the neutrophils provoked excessive IL-1β production. APaget’s disease, arthritis, periodontal disease, and chronic recurrent multifocal osteomyelitis (CRMO) are a major health Significance burden to human health. The incidence of these diseases is projected to rise due to the increase in human life expectancy and changes in diet. IL-1 therapies have shown some promise in IL-1 is a pleiotropic cytokine involved in a myriad of autoinflammatory disorders. In proline-serine-threonine phospha- alleviating disease symptoms; however, IL-1 therapies target Pstpip2cmo β both IL-1β and IL-1α, which have known distinct functions (1–3). tase-interacting protein 2 ( ) mice, IL-1 provokes Furthermore, the signaling pathways that underlie IL-1–mediated autoinflammatory osteomyelitis. Here, we demonstrated the redundant roles of Nod like receptor family, pyrin domain con- autoinflammation in these bone diseases are mostly unknown. taining 3 (NLRP3) inflammasome with caspase 8 in the process- Proline-serine-threonine phosphatase-interacting protein 2 cmo ing of IL-1β in Pstpip2 mice. Identification of redundancy (Pstpip2) gene is located on chromosome 18 in both mouse and between NLRP3 inflammasome and caspase 8 is fundamental in human. PSTPIP2 is a member of the Fes/CIP4 homology-bin/ our understanding of the inflammasomes and alternative modes amphiphysin/rvs (F-BAR) family of proteins also known as the of IL-1β regulation in osteomyelitic disease. Moreover, IL-1β pombe cdc15 homology family proteins (4). The F-bar domain signaling connects distinct compartments in promoting the dis- of PSTPIP2 has been known to interact with phosphatidyl- ease, identifying previously unidentified checkpoints that could inositol bisphosphate, whereas its C-terminal domain binds be targeted for therapeutic purposes in similar disease settings. protein tyrosine phosphatases of the PEST (a domain rich in β – Thus, our studies have unraveled the complex IL-1 regulatory proline, glutamic acid, serine, and threonine) family (5 7). network in vivo in a mouse model of osteomyelitis that will be Recent studies have shown that PSTPIP2 can also interact with useful for autoinflammatory diseases in general. inhibitory enzymes CsK and SHIP1 (8). Whereas human pa- PSTPIP2 tients with genetic mutations in the gene have not Author contributions: P.G. and T.-D.K. designed research; P.G. and A.B. performed re- been identified, missense L98P mutation in the gene Pstpip2 in search; P.G., A.B., and T.-D.K. analyzed data; and P.G. and T.-D.K. wrote the paper. mice results in severe autoinflammatory disease of the bones The authors declare no conflict of interest. that mimics CRMO in humans and thus these mice are re- This article is a PNAS Direct Submission. Pstpip2cmo – ferred to as mice (9 11). Patients with CRMO are 1To whom correspondence should be addressed. Email: thirumala-devi.kanneganti@ mostly composed of children and present with a wide range of stjude.org. symptoms ranging from bone inflammation, destruction, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. deformity (12, 13). However, the current treatment options 1073/pnas.1601636113/-/DCSupplemental.

4452–4457 | PNAS | April 19, 2016 | vol. 113 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1601636113 Downloaded by guest on September 27, 2021 cmo Finally, using bone marrow chimeras, we uncovered the complete bone disease in Pstpip2 mice, suggesting NLRP3 inflammasome IL-1β signaling loop, whereby IL-1β produced by neutrophils as the caspase-1 complex that plays a redundant role with caspase 8. (hematopoieitic compartments) is sensed by IL-1R on radio- Recent studies published from our laboratory and others have resistant cells to provoke autoinflammation and bone disease. shown that caspase 8 and caspase 1 can localize in the same inflammasome puncta containing ASC to process IL-1β (16–18). Results However, inflammasome-independent functions for caspase 8 in NLRP3 Inflammasome Plays a Redundant Role with Caspase 8 to Drive processing IL-1β have also been described (19–22). Our obser- Disease in Pstpip2cmo Mice. Whereas initial studies suggested that vation that deficiency of either caspase 1 or caspase 8 did not cmo NLRP3, Apoptosis-associated speck-like protein containing a prevent disease progression in Pstpip2 mice indicates that the card (ASC), and caspase 1 (inflammasome) were dispensable for two caspases are part of distinct and independently activated β– Pstpip2cmo complexes (Fig. 1 and ref. 2). Neutrophils are critical for disease IL-1 mediated disease in mice (1, 15), our recent cmo studies suggest that caspase 1 plays a redundant role with caspase induction in Pstpip2 mice, because the deletion of neutrophils cmo cmo 8 in promoting IL-1β secretion and disease in Pstpip2 mice can completely prevent disease in Pstpip2 mice (2). Thus, we (2). In line with these findings, we found no role for several asked whether caspase-1 and caspase-8 puncta could be visual- proteases (2) or cathepsin B and cathepsin G (Fig. S1). In- ized in neutrophils. Interestingly, we observed a significantly higher number of both caspase-1 and caspase-8 puncta in terestingly, deficiency of cathepsin C significantly delayed pro- cmo cmo Pstpip2 gression of disease in Pstpip2 mice, although most of the mice neutrophils compared with WT controls (Fig. 2). developed disease (Fig. S1). Whether cathepsin C plays a re- Whereas most of the neutrophils contained either caspase-1 or dundant role with other cathepsins or caspase 1 and caspase 8 is caspase-8 puncta alone, we also observed some cells that con- not known and will be investigated in the future. Our detailed tained both caspase-1 and caspase-8 puncta. Altogether, these data suggest that NLRP3 inflammasome plays a redundant role with a analysis of these mice showed that complete deficiency of both cmo cmo caspase-8 complex in promoting disease in Pstpip2 mice. caspase 1 and caspase 8 in Pstpip2 mice provides protection in more than 70% of the mice up to 120 d, although some mice TNF-Signaling Pathway Does Not Engage Inflammatory Caspase 8 in A B cmo demonstrate delayed clinical signs of disease (Fig. 1 and ). Pstpip2 Mice. One of the most well-established pathways that Interestingly, haploinsufficiency of caspase 8 or caspase 1 in re- lead to caspase-8 activation is the TNF-signaling pathway. En- – – Pstpip2cmo spective caspase-1 and caspase-8 deficient mice gagement of TNF receptor induces formation of the death significantly delayed progression of the disease (Fig. 1 A and B). – INFLAMMATION complex that consists of FADD caspase 8, whereby caspase 8 is IMMUNOLOGY AND To determine whether NLRP3 played a redundant role with cmo activated, ultimately resulting in apoptotic cell death. Whereas caspase 8, we generated Pstpip2 mice that lacked both NLRP3 TNF–TNF-R engagement is known to induce apoptosis, under and caspase 8. Whereas NLRP3- or caspase-8 single-deficient certain conditions caspase-8–mediated processing of IL-1β and mice developed severe disease, the mice lacking both molecules IL-18 is more prominent (20). Moreover, anti-TNF therapy has show significant protection from progression of arthritis and also been implicated as a potential therapeutic against inflammatory bone disease, suggesting a redundant role of NLRP3 with cas- bone diseases in clinics (23, 24). Thus, we proposed that the TNF- cmo pase 8 (Fig. 1 C and D). These data demonstrate that NLRP3, signaling pathway engages inflammatory caspase 8 in Pstpip2 cmo −/− akin to caspase 1, plays a redundant role with caspase 8 to provoke mice. Pstpip2 × Tnfr mice developed disease with kinetics

A B Pstpip2cmoxRipk3-/- Pstpip2cmo xCasp1-/-xCasp8+/- WT (n=5) Pstpip2cmoxRipk3-/- Pstpip2cmo (n=13) xCasp1+/-xCasp8-/- (n=12) Pstpip2cmoxRipk3-/- Pstpip2cmoxRipk3-/- xCasp1-/-xCasp8+/- (n=11) xCasp1-/-xCasp8-/- (n=28) 100 Pstpip2cmoxRipk3-/- Pstpip2cmoxRipk3-/- xCasp1+/-xCasp8-/- xCasp1-/-xCasp8-/- 50 **** *** Disease free (%) Disease free **** 0 Fig. 1. NLRP3 inflammasome plays redundant 0 306090120 roles with caspase 8 to promote osteomyelitis. Days (A and B) Pstpip2cmo, Pstpip2cmo × Rip3−/− × Casp1−/− × Casp8+/−, Pstpip2cmo × Rip3−/− × Casp1+/− × Casp8−/−, C cmo -/- − − − − − − WT (n=9) Pstpip2 x Ripk3 and Pstpip2cmo × Rip3 / × Casp1 / × Casp8 / mice -/- -/- cmo Pstpip2cmo (n=15) x Casp8 x Nlrp3 (n=17) Pstpip2cmoxRipk3-/- were generated by breeding Pstpip2 mice with D −/− × −/− × −/− Pstpip2cmo xCasp8-/-xNlrp3-/- Rip3 Casp1 Casp8 mice. Disease-free curves 100 (A) and footpad images depicting bone deformity and inflammation (B) from the indicated Pstpip2cmo strains of mice on day 150. (C and D) Pstpip2cmo × Nlrp3−/− mice were bred with Ripk3−/− × Casp8−/− 50 − − − − mice to generate Pstpip2cmo × Ripk3 / × Casp8 / × **** − − Nlrp3 / mice. Disease scores (A) and footpad images

Disease free (%) Disease free − − − − of Pstpip2cmo and Pstpip2cmo × Ripk3 / × Casp8 / × − − 0 Nlrp3 / mice on day 150 are shown. Disease curves 0306090120 were analyzed using the log-rank (Mantel–Cox) Days test. ***P < 0.001 and ****P < 0.0001.

Gurung et al. PNAS | April 19, 2016 | vol. 113 | no. 16 | 4453 Downloaded by guest on September 27, 2021 the notion that hematopoietic IL-1β is sufficient and critical CASP8 CASP1 DAPI Merged cmo A for progression of disease in Pstpip2 mice (Fig. 3 A and B). cmo In line with the phenotype observed from the Pstpip2 chi- WT meras, increased lymph node cellularity and granulocyte num- cmo bers were observed in the diseased mice (Pstpip2 >>WT cmo cmo −/− and Pstpip2 >>Pstpip2 × Il1b chimeras), compared cmo −/− with the protected mice (Pstpip2 × Il1b >>WT chimeras) (Fig. 3 C and D).

IL-1R in the Radioresistant Compartment Is Critical for Disease cmo Pstpip2cmo Observed in Pstpip2 Mice. Our data suggest that dysregu- lated IL-1β produced by the hematopoietic compartment (most probably neutrophils) (2) is important for disease progression (Fig. 3). We next asked whether autocrine IL-1β signaling of the hematopoietic cells or whether IL-1β signaling in the radioresistant compartment was important for the progression of B disease. Previous studies have shown that IL-1R deficiency 30 40 provides complete protection from disease progression in cmo cmo * * Pstpip2 mice(1,15).WhereasPstpip2 >>WT chimeras 30 developed full footpad inflammation, bone disease associated 20 with increased lymph node cellularity and granulocyte num- cmo cmo −/− 20 bers, Pstpip2 >>Pstpip2 × Il1r chimeras were completely protected suggesting the importance of IL-1R in the radioresistant 10 10 compartment (Fig. 4). To test whether hematopoieitic IL-1R was Pstpip2cmo × Percent cells with Percent cells with caspase-8 puncta important for progression of disease, we generated Caspase-1 puncta −/− Il1r >>WT chimeras. These chimeras developed disease 0 0 cmo cmo cmo similar to Pstpip2 >>WT chimeras, which is interesting, WT Pstpip2 WT Pstpip2 cmo −/− given that the Pstpip2 × Il1r mice are completely pro- Fig. 2. Caspase 1 and caspase 8 form independent complexes in Pstpip2cmo tected (Fig. 4). These data suggest that the immune cells from cmo −/− neutrophils. (A) Neutrophils were isolated from bone marrow cells of protected Pstpip2 × Il1r mice are capable of inducing Pstpip2cmo and their littermate controls by centrifugation in Percoll. Isolated disease in WT mice and that IL-1R signaling in the hematopoietic neutrophils were plated on chambered glass slides and stained with caspase-1 compartment is dispensable for disease progression. and caspase-8 antibody. The caspase-1 and caspase-8 staining in the neutrophils was visualized by immunofluorescence microscopy. DAPI was used Discussion cmo to stain the nucleus of the cells. (B) Percent of cells with caspase-1 and Our current studies demonstrate that Pstpip2 mice deficient caspase-8 puncta. Data represent means ± SEM and are representative of at ’ in both caspase 1 and caspase 8 are significantly protected from least three independent experiments. Student s t test was used to test Pstpip2cmo significance. *P < 0.05. bone disease observed in mice. Whereas single deficiency of either caspase 1 or caspase 8 does not provide protection (1, 2, 15), deficiency of both caspase 1 and caspase 8 in cmo cmo similar to Pstpip2 mice, demonstrating the dispensable role of Pstpip2 mice provided significant protection from induction of the TNF-signaling pathway in disease progression (Fig. S2A). To bone disease (2). Furthermore, the effect of either caspase 1 or further assess whether TNF-R engages caspase 8 to play a redundant caspase 8 is dose-dependent, because haplodeficiency of either cmo cmo role with caspase 1 in Pstpip2 mice, we generated Pstpip2 × of these caspases delays the progression of disease (Fig. 1A). −/− −/− Tnfr × Casp1 mice. However, double deficiency of TNF-R Recent studies have shown that caspase 1 and caspase 8 function and caspase 1 failed to provide significant protection in together in the same inflammasome complex (16–18, 25) and cmo Pstpip2 mice (Fig. S2B). Thus, we concluded from these data independently (19–21), to process IL-1β and IL-18. Deficiency that TNF-R signaling is dispensable and does not engage in- of ASC does not provide any protection from bone disease in cmo cmo flammatory caspase-8 complex in diseased Pstpip2 mice. Pstpip2 mice, suggesting that caspase 1 and caspase 8 function independently in this model of CRMO (1, 15). In support of this IL-1β Secretion by Hematopoietic Cells Is Necessary and Required for hypothesis, our microscopy data revealed distinct caspase-1 and cmo cmo Disease Progression in Pstpip2 Mice. Our early studies clearly caspase-8 puncta in neutrophils from diseased Pstpip2 mice demonstrated that IL-1β, but not IL-1α, specifically provoked that are rarely detected in the neutrophils from WT mice. cmo autoinflammatory disease in Pstpip2 mice (1). Herein, we have Caspase 1 is a prototypical caspase that is activated in the extended upon these studies to demonstrate how IL-1β provokes inflammasome complex. Our findings demonstrate that the cmo autoinflammatory disease in Pstpip2 mice. We generated bone caspase-1 complex contains NLRP3, and this complex plays a marrow chimeras to test whether IL-1β in the hematopoietic or redundant role with caspase 8 in processing IL-1β,whichis cmo cmo radioresistant compartment instigates disease in Pstpip2 mice. critical for disease induction. Generation of Pstpip2 mice cmo Pstpip2 >>WT mice developed disease, demonstrating the im- deficient in both Casp8/Casp1 and Casp8/Nlrp3 has yielded portance of the hematopoietic compartment in provoking disease. several unexpected results. Whereas we and others had pre- cmo Concurrently, WT>>Pstpip2 chimeras fail to develop bone dis- viously shown that NLRP3/caspase 1 and caspase 8 are dis- cmo ease as evident by lack of arthritis, lymph node swelling, and accu- pensable for induction of disease in Pstpip2 mice (1, 15), mulation of neutrophils (Fig. S3). We and others have previously our findings here established that both NLRP3/caspase 1 and cmo −/− shown that Pstpip2 × Il1b DKO mice are completely protected caspase 8 are critical for progression of disease. Our studies also cmo from inflammation and disease (1, 14, 15). Interestingly, Pstpip2 >> exclude the TNF-signaling pathway in engaging caspase 8; how- cmo −/− Pstpip2 × Il1b chimeras developed disease, suggesting IL-1β ever, other TNF-family members such as CD95 and TRAIL might produced by radioresistant cells is dispensable for disease pro- be involved and will be investigated in future studies (20, 26). cmo −/− gression (Fig. 3 A and B). Conversely, Pstpip2 × Il1b >>WT Caspase 8 has also been shown to be activated in the Dectin–Syk– mice did not develop any significant disease, further strengthening CARD9 complex and function as an IL-1β–processing protease

4454 | www.pnas.org/cgi/doi/10.1073/pnas.1601636113 Gurung et al. Downloaded by guest on September 27, 2021 A Pstpip2cmo >> WT (n=8) C 40 **** cmo -/-

Pstpip2 xIl1b >> WT (n=10) ) 6 Pstpip2cmo>>Pstpip2cmoxIl1b-/-(n=8) 30 100 **** 20

50 (x10 PLN Total 0

**** -/- -/- Disease free (%) Disease free cmo >>

0 xIl1b xIl1b 0 255075100 cmo >>WT cmo cmo Pstpip2

Days post chimerism >>WT Pstpip2 Pstpip2 B D Pstpip2 cmo cmo cmo 25 Pstpip2 Pstpip2 Pstpip2 >> **** -/- cmo -/- >>WT xIl1b >>WT Pstpip2 xIl1b per

) 20 + 4 15 **** Fig. 3. IL-1β secretion by hematopoietic cells provokes disease in Pstpip2cmo mice. (A) Disease incidence in Pstpip2cmo>>WT,

CD11b cmo −/− cmo cmo −/− + 10 Pstpip2 × Il1b >>WT,andPstpip2 >>Pstpip2 × Il1b

PLN (x10 PLN >> 5 mice (donor recipient). (B) Footpad images of the indicated

# Gr1 chimeras harvested on day 100 postchimerism showing the extent 0 of footpad inflammation and bone disease. Total number of popli- + + -/- -/- teal lymph node cells (C)andGr1CD11b neutrophils within the

cmo cmo −/− >> popliteal lymph nodes (D) from the indicated Pstpip2 /Il1b chi-

xIl1b xIl1b ± =

cmo meras. Data in C and D represent means SEM with n 8for INFLAMMATION cmo cmo −/− IMMUNOLOGY AND >>WT cmo cmo Pstpip2 >>WT, n = 10 for Pstpip2 × Il1b >>WT,andn = 8for − −

Pstpip2 cmo cmo / >>WT Pstpip2 >>Pstpip2 × Il1b mice. Disease curves were analyzed using the log-rank (Mantel–Cox) test. The Mann–Whitney test was Pstpip2 < Pstpip2 Pstpip2 used to determine significance between groups. ****P 0.0001.

cmo (21), and it is possible that the Dectin–Syk–CARD9 pathway might Pstpip2 hematopoietic cells are critical and sufficient for cmo engage caspase 8 in Pstpip2 mice. Our study highlights the induction of disease (1). Given that IL-1β is critical for induction complexity of these autoinflammatory bone diseases, where mul- of disease (1, 15), we asked whether hematopoietic cells were the tiple pathways could be working in unison or independently. major source of IL-1β. Our studies demonstrated that IL-1β

AC** cmo 25 **** Pstpip2 >> WT (n=6) ) 6 Pstpip2cmoxIl1r-/- >> WT (n=12) 20 cmo cmo -/- Pstpip2 >>Pstpip2 xIl1r (n=7) 15 100 10 5 Total PLN (x10 PLN Total 50 0 -/- -/- cmo

**** >> Disease free (%) Disease free xIl1r xIl1r cmo cmo cmo

0 >>WT

0 25 50 75 100 Pstpip2 >>WT Days post chimerism Pstpip2 Pstpip2 B D Pstpip2 15 *** Pstpip2cmo Pstpip2cmo Pstpip2cmo>> **** Fig. 4. IL-1β signaling through IL-1R in the radioresistant cells is per cmo ) -/- cmo -/- + necessary for induction of disease in Pstpip2 mice. (A)Disease >>WT xIl1r >>WT Pstpip2 xIl1r 4 − − 10 incidence in Pstpip2cmo>>WT, Pstpip2cmo × Il1r / >>WT,and − − Pstpip2cmo>>Pstpip2cmo × Il1r / mice (donor>>recipient). (B)Footpad CD11b + 5 images of the indicated chimeras harvested on day 100 postchimerism PLN (x10 PLN showing the extent of footpad inflammation and bone disease. Total + +

# Gr1 numbers of popliteal lymph node cells (C) and Gr1 CD11b neu- 0 trophils within the popliteal lymph nodes (D) from the indicated

-/- -/- − − Pstpip2cmo/Il1r / chimeras are shown. Data in C and D represent cmo >> ± = cmo>> = cmo × xIl1r xIl1r means SEM with n 6forPstpip2 WT, n 12 for Pstpip2 cmo −/−>> = cmo>> cmo × −/−

cmo cmo Il1r WT,andn 7forPstpip2 Pstpip2 Il1r mice. >>WT – Pstpip2 >>WT Disease curves were analyzed using the log-rank (Mantel Cox) test. The Mann–Whitney test was used to determine significance Pstpip2 < < < Pstpip2 Pstpip2 between groups. **P 0.01, ***P 0.001, and ****P 0.0001.

Gurung et al. PNAS | April 19, 2016 | vol. 113 | no. 16 | 4455 Downloaded by guest on September 27, 2021 secreted by the hematopoietic compartment is necessary and sufficient for disease induction and progression, and that IL-1β produced by the radioresistant compartment is dispensable. Whereas our chimeras only demonstrate whether hematopoietic or the radioresistant compartment are important for IL-1β secretion, based on previous studies (2), it could be posited that the major IL-1β–producing cells are the neutrophils. Indeed, anti- cmo Ly6G Ab treatment (that depletes neutrophils) of Pstpip2 mice prevents induction of bone disease (2). Although it is possible that myeloid cells as well as other immune cells might partake in the induction and propagation of disease, neutrophils are essential cmo instigators of the disease in Pstpip2 mice. IL-1β is a pleiotropic cytokine that affects target cells to produce several important cytokines and chemokines that ultimately contribute to the induction of bone disease. Importantly, cytokines such as TNF-α, MCP-1, and M-CSF, which are highly up- cmo regulated in Pstpip2 mice, are all at basal level in age-matched cmo −/− Pstpip2 × Il1b mice (1). Thus, IL-1β is an apical cytokine that signals through target cells to amplify inflammatory milieu cmo and provoke autoinflammatory bone disease in Pstpip2 mice. cmo −/− We generated chimeras from Pstpip2 and Il1r mice to investigate the compartment that responds to IL-1β signal to in- cmo stigate disease in Pstpip2 mice. Interestingly, IL-1β signaling in radioresistant cells, but not hematopoietic cells, is critical for cmo disease induction in Pstpip2 mice. Adding to this observation, cmo −/− hematopoietic cells from protected Pstpip2 × Il1r mice can Fig. 5. Model showing IL-1β–mediated inflammation and bone disease in cmo −/− cmo cmo induce disease in WT recipients (Pstpip2 × Il1r >>WT chi- Pstpip2 mice. Pstpip2 mutation in hematopoietic cells results in induction of distinct caspase-1 and caspase-8 complexes (most probably within the neutro- mera develop full disease), further confirming that autocrine IL- β phils), i.e., PSTPIP2 might actively suppress spontaneous formation of these 1 signaling in hematopoietic cells is not necessary for disease complexes. Whereas caspase 1 is activated within the NLRP3 inflammasome induction. Altogether, the chimera data generated in this study complex, caspase 8 is activated in a yet unknown complex. Regardless, these demonstrate the directionality of IL-1β signaling required for caspases work in a redundant manner to cleave pro–IL-1β into their mature the induction of disease. Future studies will investigate the cell bioactive forms. Once secreted by the hematopoietic cells, bioactive IL-1β is sensed populations in the radioresistant compartments that respond to IL- by IL-1R on radioresistant cells to promote inflammation and bone disease. cmo 1β signals to instigate the bone disease in Pstpip2 mice. Chronic multifocal osteomyelitis is a severe autoinflammatory Percoll (GE Healthcare) gradient after a 30-min centrifugation at 1,300 × g for bone disease commonly observed in children and adolescents 30 min at 4 °C. with severe consequences on the affected individuals resulting in Neutrophils were then plated onto four-chambered glass slides pretreated permanent bone deformity (12). Our study highlights the complex with poly-L-lysine. Neutrophils were then fixed in 4% (vol/vol) paraformaldehyde redundancy between NLRP3 inflammasome and caspase 8 in for 15 min at room temperature, followed by nonspecific blocking in goat se- processing IL-1β and instigating the debilitating bone disease in rum (DAKO) containing 0.1% saponin (Sigma). Following blocking, neutrophils cmo Pstpip2 mice (a mouse model for chronic multifocal osteomy- were gently washed three times and stained with either mouse Casp1 (AG-20B- elitis) (Fig. 5). We further demonstrate that IL-1β produced by 0042-C100, Adipogen) or rat caspase-8 (1G12, Enzo) antibody for 1 h at 37 °C. hematopoietic cells signals through radioresistant cells to induce Neutrophils were gently washed three times again and stained with sec- cmo inflammation and bone disease in Pstpip2 mice. Our studies ondary antibodies (goat anti-rat Alexa Fluor 488, goat anti-mouse Cy3 from provide a previously unidentified description of upstream mole- Life Technologies) for 40 min at 37 °C. Neutrophils were then washed four cules as well as potential cellular targets that can be intercepted in times in warm PBS, air dried, and mounted with DAPI. The images were the IL-1β pathway to treat this debilitating bone disease. analyzed and imaged using a Nikon C2 confocal microscope. For each slide, ∼50 cells were counted three independent times (different regions) and cells Methods positive for caspase-1 and caspase-8 puncta of these 50 cells were noted and represented as percentages. Three to four independent slides were used for Mice and Breeding. All mice were kept in a specific pathogen-free environment each experiment. For statistical analysis, an average percentage of caspase-1 at the St. Jude Children’s Research Hospital Animal Facility. Pstpip2cmo mice or caspase-8 puncta from each slide was used. were previously described and purchased from The Jackson Laboratory and

were on the BALB/c background (9). All other mutant mice were on C57BL/6 cmo cmo −/− − − × background or backcrossed to C57BL/6 mice for at least 10 generations. Nlrp3 / Generation of Chimeras. Four- to six-week-old WT, Pstpip2 , Pstpip2 Il1b , cmo × −/− (27), Casp1−/− (27), Il1b−/− (28), Il1r−/− (29), Ripk3−/−Casp8−/− (30), Ctsb−/− (31), and Pstpip2 Il1r mice were lethally irradiated with 900 rads. Six to − − − − − − Ctsc / (32), Ctsg / (33), and Tnfr / (34) mice have been previously described. eight hours after lethal irradiation, these mice received bone marrow cells cmo cmo × −/− cmo × −/− Pstpip2cmo mice (BALB/c background) were crossed with C57BL/6 mice to from donor WT, Pstpip2 , Pstpip2 Il1b ,andPstpip2 Il1r generate mixed background Pstpip2cmo mice for chimera experiments. mice. Briefly, bone marrow cells were harvested from tibias and femurs Pstpip2cmo mice were crossed with the indicated genetic KO mice to test the of mice and passed through 18-gauze needles to generate single cell sus- 6 role of a particular gene in bone disease. Littermate controls were used to pension. After filtering through a 40-μm cell strainer, about 5 × 10 bone determine whether genetic deletions influenced osteomyelitic bone disease marrow cells were transferred via retroorbital sinus into the lethally irra- development. Animal studies were conducted under protocols approved by diated mice. These mice were then followed twice weekly for signs of St. Jude Children’s Research Hospital. footpad inflammation and osteomyelitic bone disease.

Neutrophil Isolation and Confocal Imaging. Bone marrow was flushed from the Mice Harvest and Flow Cytometry Analysis. Mice were killed using CO2 in- tibias and femurs of WT and diseased Pstpip2cmo mice. Bone marrow cells halation according to the approved guidelines from St. Jude Children’s were then passed through 18-gauge needles at least three times to make a Research Hospital. Following killing, popliteal lymph nodes and spleen single cell suspension. These cells were filtered through a 70-μm cell strainer were harvested. Single cell suspension of cells from lymph node and spleen to get rid of clumps, and neutrophils were purified from the interface of 62.5% were made as described previously (35). Cells were then stained for neutrophils

4456 | www.pnas.org/cgi/doi/10.1073/pnas.1601636113 Gurung et al. Downloaded by guest on September 27, 2021 [anti-Ly6G Percp5.5 Ab (Biolegend) and anti-CD11b eFlour450 Ab (eBioscience)] ACKNOWLEDGMENTS. We thank Drs. Shizuo Akira, Gabriel Nunez, Richard andanalyzedusingtheBDFacsCaliburandFlowJosoftware. Flavell, John Bertin, David Chaplin, Douglas Green, Thomas Reinheckel, and Christine Pham for generous supply of mutant mice and Drs. Deepika Sharma, David Place, and Farrah Phillips for their help with editing Statistical Analysis. All data are represented as mean ± SEM and all experiments the manuscript. P.G. is a postdoctoral fellow supported by a Paul Barrett were repeated at least two to three times before being reported. Survival curves Endowed Fellowship from St. Jude Children’s Research Hospital. T.-D.K. is – ’ were analyzed using the log-rank (Mantel Cox) test. Two-tailed Student s t test supported by the National Institutes of Health (Grants AR056296, and Mann–Whitney u test were used to compare statistical significance between CA163507, and AI101935) and the American Lebanese Syrian Associated two groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Charities.

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