Cleaved Cochlin Sequesters Pseudomonas Aeruginosa and Activates Innate Immunity in the Inner Ear

Article

Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear

Graphical Abstract Authors Jinsei Jung, Jee Eun Yoo, Young Ho Choe, ..., Joo-Heon Yoon, Young-Min Hyun, Jae Young Choi

Correspondence [email protected] (Y.-M.H.), [email protected] (J.Y.C.)

In Brief Jung et al. show that the cleaved cochlin LCCL domain enhances innate immune responses in the inner ear by aggregating inﬁltrated bacteria and recruiting innate immune cells. This spatiotemporally protective function of LCCL protects hearing function in the organ of Corti against effects of bacterial invasion in the inner ear.

Highlights d Cleaved inner ear cochlin LCCL secretes to perilymph space post-bacterial infection d LCCL induces bacterial aggregation in the scala tympani d Spatiotemporal innate immune response by LCCL protects the sensory organ of Corti

À À d LCCL rescues Coch / mouse post-Pseudomonas inner ear infection hearing loss

Jung et al., 2019, Cell Host & Microbe 25, 1–13 April 10, 2019 ª 2019 Elsevier Inc. https://doi.org/10.1016/j.chom.2019.02.001 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001 Cell Host & Microbe Article

Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear

Jinsei Jung,1,2,7 Jee Eun Yoo,1,2,7 Young Ho Choe,3,4 Sang Chul Park,1 Hyun Jae Lee,1,2 Hack June Lee,1,2 Byunghwa Noh,1,2 Sung Huhn Kim,1,2 Gyeong-Yi Kang,3,4 Kang-Mu Lee,5 Sang Sun Yoon,4,5 Dong Su Jang,6 Joo-Heon Yoon,1,2 Young-Min Hyun,3,4,* and Jae Young Choi1,2,8,* 1Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, South Korea 2Airway Mucus Institute, Yonsei University College of Medicine, Seoul, South Korea 3Department of Anatomy, Yonsei University College of Medicine, Seoul, South Korea 4Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea 5Department of Microbiology and Immunology, Yonsei University College of Medicine, Seoul, South Korea 6Department of Sculpture, Hongik University, Seoul, South Korea 7These authors contributed equally 8Lead Contact *Correspondence: [email protected] (Y.-M.H.), [email protected] (J.Y.C.) https://doi.org/10.1016/j.chom.2019.02.001

SUMMARY terial and viral invasion along with endogenous host-derived molecules associated with acoustic overstimulation (Cai et al., In the inner ear, endolymph fluid surrounds the or- 2014; Vethanayagam et al., 2016). The innate immune response gan of Corti, which is important for auditory func- eliminates apoptosis-derived antigens from endogenous tis- tion; notably, even slight environmental changes sues by toll-like receptor 4-myeloid differentiation factor 2 mediated by trauma or infection can have signifi- (TLR4-MD2) signaling. The rapid recruitment of phagocytes, cant consequences. However, it is unclear how such as macrophages and neutrophils, during the innate im- the immune response is modulated in these tissues. mune response is important for promptly eliminating invaded pathogens and induces the inflammatory changes responsible Here, we report the local immune surveillance role Limulus for the subsequent architectural damage of the organ of Corti of cleaved cochlin LCCL ( factor C, Cochlin, (Woo et al., 2015). Thus, the innate immune response requires Pseudomonas aeruginosa and Lgl1) during infection fine regulation and spatiotemporal control to efficiently elimi- in the cochlea. Upon infection, the LCCL domain is nate pathogens while avoiding damage to the sensory hair cells cleaved from cochlin and secreted into the peri- and preserving the endolymph space harboring the organ of lymph. This cleaved fragment sequesters infiltrating Corti. However, the mechanisms modulating this tight control bacteria in the scala tympani and subsequently re- are largely unknown. cruits resident immune cells to eliminate the bacte- Cochlin, encoded by the COCH gene, is abundantly ex- ria. Importantly, hearing loss in a cochlin knockout pressed in the inner ear, eye, and spleen (Robertson et al., mouse model is remedied by treatment with a co- 1994). Its protein structure consists of a Limulus factor C, Co- chlin LCCL peptide. These findings suggest cleaved chlin, and Lgl1 (LCCL) domain and two von Willebrand factor A-like (vWFA) domains that presumably bind to extracellular ma- cochlin LCCL constitutes a critical factor in innate trix components. The LCCL domain has strong homology to immunity and auditory function and may be a po- Limulus factor C, an endotoxin-sensitive serine proteinase that tential therapeutic target to treat chronic otitis me- is involved in the immune response in the horseshoe crab, dia-induced hearing loss. possibly functioning as an anti-bacterial peptide (Muta et al., 1991; Robertson et al., 1998). Notably, mutations in COCH are linked to inherited progressive sensorineural hearing loss INTRODUCTION (DFNA9), indicating that normal cochlin expression in the inner ear is critical for maintaining auditory function (Robertson Sensory organs, such as the inner ear and the eyes, are crucial et al., 1998). In addition, accumulated cochlin in the eye is asso- for auditory and visual perception. In these highly differentiated ciated with glaucoma (Bhattacharya et al., 2005b). As the inner and sensitive organs, even minimal damage resulting from ear and eye are similar with regard to the presence of a fluid infection or trauma may irreversibly deteriorate hearing or visual compartment (i.e., the perilymph space and anterior eye cham- function. For example, the endotoxin generated during a middle ber, respectively) that expresses abundant cochlin (Bhatta- ear bacterial infection can compromise the round window charya et al., 2005b; Ikezono et al., 2004), it is speculated that membrane, leading to inflammatory damage of the cochlear cochlin may have an as-yet undefined physiological role in sen- structures and sensory cells and sensorineural hearing loss sory organs. (Engel et al., 1995; Kawauchi et al., 1989; Moon et al., 2006). It has been reported that cochlin in the spleen promotes sys- In the cochlea, the immune response protects the sensory or- temic innate immunity against bacterial infection (Py et al., gan of Corti, an essential inner ear compartment, against bac- 2013). Indeed, the LCCL domain is cleaved from spleen-derived

Cell Host & Microbe 25, 1–13, April 10, 2019 ª 2019 Elsevier Inc. 1 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

Figure 1. Cochlin in the Cochlea Protects Hearing Function during Bacterial Infection (A) Overview of the cochlea. The mammalian cochlea consists of the scala media that harbors the sensory epithelial structure organ of the Corti (filled with endolymph) and the scala tympani/vestibuli (filled with perilymph). The lateral cochlear wall is surrounded by spiral ligaments consisting of fibrocytes and various types of collagen. (B) Cochlin protein (green) is distinctly expressed in the spiral ligaments in the lateral wall and many supporting cells along the basement membrane of the cochlea and crista ampullaris but is not expressed in cochlin knockout mice (Coch/). Myosin7A expression (red) indicates cochlear hair cells. Nuclei are stained in blue (DAPI). Cochlin mRNA is dominantly expressed in the spiral ligament at 8 weeks of age (lower panels). Scale bars, 50 mm. (C–E) Representative traces of auditory brainstem response (ABR) tests (C) performed in both Coch+/+ and Coch/ mice at 4 weeks of age with and without P. aeruginosa infection. The response to click stimuli (n = 13 mice in each group) (D) and tone-burst stimuli at 4000, 8000, 16,000, and 32,000 Hz (n = 4 mice in each group) (E) was evaluated during the ABR tests. **p < 0.01, *p < 0.05 by two-way ANOVA with Bonferroni correction for multiple comparisons. See also Figure S1. cochlin and secreted into the blood; then, it accumulates in the ment and interleukin (IL)-1b/IL-6 cytokine secretion. These inflammatory lesions in other bacteria-infected tissues such as findings demonstrate a surveillance role for cochlin in the inner the lung, promoting the innate immune response (Py et al., ear and show that the cleaved LCCL domain helps to preserve 2013). Although this previous study again indicates an essential critical sensory organs, including the organ of Corti, and audi- function for cochlin in the innate immune response, how the tory function. cleaved fragment increases local pro-inflammatory cytokine secretion and recruits innate immune cells is not well under- RESULTS stood. In fact, the LCCL domain itself does not appear to exert bactericidal effects by lipopolysaccharide (LPS) binding and Cochlin Protects Hearing during Infection cannot promote cytokine secretion from macrophages (Py The cochlea consists of the scala media (harboring the organ of et al., 2013), suggesting that other mechanisms are involved. Corti surrounded by endolymph) and the perilymph-filled scala In the present study, we investigated the role of cochlin in tympani and vestibuli (Figure 1A). In wild-type mice, cochlin pro- the inner ear during Pseudomonas aeruginosa infection of tein is primarily expressed in the spiral ligament, limbus, and the perilymph, which is the initial site where pathogenic bacte- crista ampullaris of the semi-circular canals at 8 weeks of age ria usually enter through the round window. Our analysis also (Figure 1B, upper panel). Cochlin mRNA was also abundantly ex- explored the presence of cleaved LCCL domain in the peri- pressed in the inner ear at this stage (Figure 1B, lower panel). To lymph space, aggregation of pathogens, and subsequent bac- investigate cochlin function in the inner ear, we compared the terial elimination, focusing on neutrophil and monocyte recruit- auditory thresholds of wild-type and Coch knockout (Coch/)

2 Cell Host & Microbe 25, 1–13, April 10, 2019 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

Figure 2. LCCL Cleavage in the Inner Ear Mediates Cochlear Immune Response and Sensory Organ Preservation (A) In ex vivo cochlea explants, P. aeruginosa infection for 1 day induced cochlin cleavage, detected by N- and C-terminal antibodies. In perilymph from the scala tympani (collected by fine needle aspiration from the explanted cochlea), only the cleaved LCCL domain form was identified and the amount increased after bacterial infection. As a positive control of cochlin expression, heterologously expressed cochlin in HEK293 cells was loaded in the first and second lanes with different protein dose (H, high dose, 10 mg; L, low dose, 3 mg). (B) LCCL peptide (2.5 mL of 100 mg/mL) was simultaneously treated with in vivo P. aeruginosa inoculation in the Coch/ ear for 1 day and acoustic threshold shift was measured in ABR test (n = 6 mice in each group). The scale out threshold indicates R100 dB. Statistical comparison was performed between P. aeruginosa- infected Coch/ groups with and without the LCCL domain. **p < 0.01 by two-way ANOVA with Bonferroni correction for multiple comparisons. (C) In ex vivo cochlear explants treated with P. aeruginosa for 1 day, bacterial growth was greater in Coch/ than Coch+/+ mice given that the color density of the well with Coch+/+ cochlea was more turbid. After serial dilution of P. aeruginosa on agar, the bacterial count of each group was measured (n = 9 mice in each group). ***p < 0.001 by Student’s t test. (D) One day after P. aeruginosa infection in the ex vivo cochlea, pro-inflammatory cytokines (IL-1b, IL-6, MCP-1, KC, and MIP-2) were measured by ELISA (n = 4–8 mice in each group). *p < 0.05; ns, not significant by two-way ANOVA with Bonferroni correction for multiple comparisons.

(E) Effect of 50 mM matrix metalloproteinase III inhibitor (MMPinh) addition into the culture media on IL-1b and IL-6 expression after P. aeruginosa infection in the ex vivo cochlea of Coch+/+ mice (n = 9 mice in each group). ***p < 0.001 by two-way ANOVA with Bonferroni correction for multiple comparisons. See also Figure S2. mice over time (Figure S1). However, the hearing thresholds did LCCL Cleavage from Cochlin Mediates Auditory not differ signiﬁcantly between these mice. Protection Although our data indicated that cochlin may not be involved We next investigated the possible mechanism of hearing preser- in normal auditory function, we also investigated its role during vation, starting with cleavage of the LCCL domain. Because co- bacterial infection. We introduced P. aeruginosa PAO1 strain chlin is also expressed in other tissues (i.e., the spleen and eyes), into the inner ear via middle ear inoculation and evaluated the we performed immunoblots from ex vivo explants of the inner ear effect of bacterial infection on auditory function in the presence to rule out the effect of cochlin from other organs. Only full-length and absence of cochlin. Hearing thresholds were well pre- cochlin (60 kDa) was identiﬁed under basal conditions (Fig- served in wild-type mice even post-PAO1 infection, whereas ure 2A). However, during PAO1 infection, cochlea-derived co- those in Coch/ mice were much higher after PAO1 infection chlin was cleaved into 8 and 18 kDa fragments, corresponding (Figures 1C–1E), indicating that cochlin preserves auditory to the unglycosylated and glycosylated LCCL domain, respec- function. tively, and secreted (Figures S2A and S2B). These molecular

Cell Host & Microbe 25, 1–13, April 10, 2019 3 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

bands were not detected in Coch/ mice. Moreover, in vivo Secreted LCCL Segregates Invading Bacteria inoculation of PAO1 into the cochlea also resulted in LCCL cleav- It is still unclear how cochlin-derived LCCL mediates innate image and secretion (Figure S2C). Cleavage of cochlin in the co- munity and hearing preservation during inner ear bacterial infec- chlea appeared to be mediated by matrix metalloproteinase tion, particularly as LCCL has not been directly linked to known (MMP) as this process was sensitive to MMP III inhibitor, which anti-bactericidal processes. After cleavage in the cochlea, the is consistent with a previous report in the spleen (Figure S2D) LCCL fragment secreted into scala tympani was co-localized (Py et al., 2013). Cochlin cleavage was also dependent on aggre- with the invading PAO1 bacteria (Figure 3). As the co-localized canase 1 and 2, which are encoded by a disintegrin and metal- cochlin was not stained with C-terminal antibody against cochlin loproteinase with thrombospondin motifs (Adamts) 4 and 5, (Figure S3A) and only the N-terminal LCCL domain was identified respectively (Figure S2E). Adamts4 mRNA expression was in the perilymph by western blot (Figure 2A), we considered that equivalently robustly increased in the cochlea of wild-type and the identified cochlin in the scala tympani mainly comprised the Coch/ mice post-PAO1 infection (Figure S2F). In contrast, LCCL domain rather than full-length cochlin. Moreover, the Adamts5 expression showed no change, indicating that aggre- PAO1 bacteria were limited to the scala tympani compartment canase 1 mainly functions in the condition of bacterial infection in wild-type mice but were dispersed throughout the cochlea, in the ear. Moreover, when exogenous LCCL was added during including in the organ of Corti, in the Coch/ mice (Figure 3, or 6 h after PAO1 inoculation into the Coch/ cochlea, the hear- magnified inset). This bacterial dispersion likely mediates the ing thresholds were significantly lower than those of the PAO1- damage observed in the organ of Corti as well as the inner and infected group (Figures 2B and S2G). Taken together, these outer hair cells of Coch/ mice compared to the well-preserved data demonstrate that cochlin expressed in the inner ear is func- tissues in wild-type mice (Figure S3B). These findings suggest tionally cleaved by aggrecanase 1 during bacterial infection and that after cleavage and then secretion into the scala tympani, the N-terminal LCCL is secreted into the scala tympani. Further- the cochlin-derived LCCL domain sequesters the bacteria in more, LCCL cleavage and secretion are essential for the protec- this compartment and inhibits spreading into the scala media, tion of auditory function during bacterial infection. thus protecting the organ of Corti.

Cochlin Mediates a Cochlear Immune Response LCCL Recruits Neutrophils and Macrophages Next, we counted the number of bacteria in the cochlear ex- Next, we investigated the role of the LCCL fragment in recruiting plants of wild-type and Coch/ mice after PAO1 inoculation. neutrophils and macrophages involved in innate immunity. To Bacterial growth in the inner ears of Coch/ mice was signifi- visualize these effects, we generated wild-type and Coch/ cantly higher than that in wild-type mice (Figure 2C), suggesting mice that heterozygously expressed enhanced green fluores- that cochlin in the inner ear plays a local anti-bacterial role. cent protein (GFP) in the lysozyme M gene (LysM-GFP+/), Thus, we evaluated various anti-bacterial and innate immu- thus allowing the identification of neutrophil and monocyte line- nity-related factors such as TLR4 in the cochlea pre- age cells (mainly neutrophils). Neutrophils were exclusively pre- and post-PAO1 infection. TLR4 mRNA expression did not sent in the peri-modiolar vessels and stria vascularis under basal significantly differ between the wild-type and Coch/ mice conditions in both the wild-type and Coch/ mice (Figure 4, top (Figure S2H). Anti-bacterial peptides such as b-defensin 3, two rows). However, these immune cells were robustly recruited b-defensin 4, Reg3g, and S100A8 did not differ between into the scala tympani after PAO1 infection in the wild-type mice wild-type and Coch/ mice post-PAO1 infection (Figure S2I). (Figure 4, third row). Notably, the neutrophils were dispersed We also measured the expression of various cytokines involved throughout the cochlea, infiltrating the organ of Corti, modiolus, in the innate immune response. To discriminate between the and lateral wall, in the PAO1-infected Coch/ mice (Figure 4, local and systemic immune responses, we measured cytokine bottom row). In the 3D structural images of the cleared cochlea expression in the culture media of explanted cochlea from by the tissue clearing method (Chung et al., 2013)(Figure 5A), both Coch+/+ and Coch/ mice pre- and post-PAO1 infection. we also observed PAO1-infection-induced clusters of neutrophil IL-1b and IL-6 expression in the media was significantly higher in the scala tympani (t) of wild-type (yellow arrowheads), but in the explants from PAO1-infected wild-type mice compared not Coch/ mice (Figure 5B; Videos S1 and S2). Because the to that in the other three tested groups (Figure 2D). Moreover, 3D tissue clearing imaging was acquired from a 5 mm gap in IL-1b and IL-6 expression was also significantly increased the z-stack slice, single immune cells with GFP signal were in vivo in the PAO1-inoculated Coch+/+ mice (Figure S2J). How- partially visualized in Coch/ mice (orange arrowheads). The ever, the levels of other cytokines (MCP-1, KC, and MIP-2) 3D tissue clearing images of cochlea revealed significantly were not affected by the presence of cochlin in the inner ear higher neutrophil counts in wild-type than Coch/ mice (Fig- (Figure 2D), suggesting that their regulation may be multifacto- ure 5C). To more quantitatively compare the number of recruited rial. These results demonstrate that cochlin expression is neutrophils and macrophages in the cochlea, we isolated these required for the local upregulation and increased secretion of cells by fluorescence-activated cell sorting (FACS; Figure S5A). IL-1b and IL-6. These changes in the wild-type PAO1-infected Under basal conditions, the numbers of macrophages and neu- mice were completely abolished in vivo and ex vivo when trophils were 0.2% and 5.4% of the total number of whole treated with MMP III inhibitor (Figure 2E). Taken together, these single cells from the cochlea, respectively (Figure S5B). The data confirm that cochlin cleavage and LCCL secretion are basal populations of these immune cells did not differ between crucial processes to induce local cytokine expression and Coch+/+ and Coch/ mice; however, a significantly higher num- the innate immune response during bacterial infection in the ber was recruited in Coch+/+ than in Coch/ mice post-PAO1 inner ear. infection (Figure 5D). We also counted the number of neutrophils

4 Cell Host & Microbe 25, 1–13, April 10, 2019 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

Figure 3. Cochlin Promotes Bacterial Aggregation in the Scala Tympani Representative immunostaining images of the Coch+/+ and Coch/ cochlea infected with GFP-tagged P. aeruginosa (green) 1 day after in vivo infection showing bacterial segregation in the scala tympani (t) of Coch+/+ mice, while being dispersed in the scala tympani to scala media (m), scala vestibuli (v), and spiral ganglion in the Coch/ mice. Magnified insets show the bacterial infected organ of Corti. The Reissner’s membrane between the scala media and vestibuli was distended (white arrowheads) and the scala media was dilated. Note that the aggregated GFP-PAO1 (green) co-localized with secreted cochlin (red, anti-N-terminal cochlin antibody) in Coch+/+ mice. Blue, nuclei/DAPI. Scale bars, 100 mm. See also Figure S3. and monocytes, respectively, in the blood and the bone marrow phages (white arrowheads in Figure 5E, magnified in the upper from both wild-type and Coch/ mice under basal conditions right inset). The infiltrated round-shaped monocytes (or imma- without bacterial infection. Numbers of these two cell popula- ture macrophages) in the cochlea were also identified with tions in these tissues did not differ between the two mouse CD115 antibody, being present dominantly in wild-type mice strains (Figure S5C). These data confirmed that innate immune (Figure S5D). In addition, I-A-/I-E-positive round-shaped den- cell populations of the bone marrow and blood in Coch/ dritic cells were exclusively observed in wild-type mice after mice are comparable to those of wild-type mice. PAO1 infection (Figure S5E). Therefore, it appears that not only In fact, F4/80-positive macrophages were found to intensively neutrophils but also immature macrophages (or monocytes) infiltrate from the spiral ligament vessels and spiral ganglion in and dendritic cells are recruited depending on the presence of the wild-type, but not Coch/, mice (yellow arrowheads in Fig- cochlin in the cochlea. ure 5E, magnified in the upper right inset). These infiltrated imma- Cytokine expression was also localized to particular areas of ture macrophages also appeared to be morphologically distinct the cochlea during bacterial infection. Under basal conditions, from resident macrophages in the cochlea, being round or ovoid IL-1b, for example, was localized in the cells along Reissner’s shape, compared to the dendritic shape of the resident macro- membrane, the spiral prominence, and spiral ganglion in both

Cell Host & Microbe 25, 1–13, April 10, 2019 5 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

Figure 4. Cochlin Regulates Neutrophil Inﬁltration in the Cochlea during In Vivo Infection Representative immunostaining images showing the presence of neutrophils (tagged with GFP in LysM+/ mice) in the stria vascularis of the lateral wall and peri- modiolar vessels (yellow and green crops in Coch+/+ and Coch/ culture media) in uninfected cochlea regardless of cochlin expression. However, after P. aeruginosa infection, neutrophils mainly inﬁltrated into the scala tympani and basilar membrane in the Coch+/+ mice but were widely dispersed throughout the cochlea in Coch/ mice. Extravasated neutrophils were robustly located in the organ of Corti (green crop in Coch/-Pseudomonas)inCoch/ mice, subsequently damaging sensory hair cells (white arrowheads). Blue, nuclei/DAPI; red, cochlin; green, GFP. See also Figure S4.

Coch+/+ and Coch/ mice. After PAO1 infection in Coch+/+ mice, monocyte recruitment and inducing inflammatory cytokine IL-1b expression was strongly increased in the spiral ligament, secretion, while protecting auditory function by sequestering stria vascularis, inner limbus, spiral ganglion, and supporting the bacteria in the scala tympani. It is unclear how this LCCL cells around hair cells, thus surrounding the scala media space fragment performs these functions, particularly as PAO1 bac- and preserving the hair cells (Figure S4, third row). In contrast, terial viability is not affected by recombinant LCCL peptide IL-1b expression in Coch/ mice post-PAO1 infection was (1 mg/mL) treatment in vitro (Figure 6A).Furthermore,the dispersed throughout the cochlea and highly expressed around growth rates of GFP-expressing PAO1 grown in agar plates the damaged basilar membrane, hair cells, and scala vestibuli, in the presence and absence of LCCL did not significantly following a similar expression pattern as that of the neutrophils differ (Figure 6B). However, when the PAO1 bacteria were and monocytes (Figure S4, bottom row). These findings indicate grown on glass coverslips with and without LCCL, their growth that cochlin has an essential role in enhancing the expression of patterns distinctly differed, being more globularly colonized inflammatory cells and cytokines in the restricted scala tympani and aggregated after LCCL addition. Indeed, the average space where the bacterial pathogens are aggregated. GFP signal in the LCCL-treated PAO1 bacteria was weaker than that of the control owing to the conglomerated bacteria LCCL Physically Interacts with Bacteria (Figures 6C and 6D; Video S3). Our data suggest that cleaved cochlin, the LCCL fragment, In addition, the aggregated PAO1 co-localized with LCCL, modulates the immune response, including neutrophil and indicating the possibility of direct interaction (red color in

6 Cell Host & Microbe 25, 1–13, April 10, 2019 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

Figure 5. Activated and Secreted Cochlin Recruits Neutrophils and Macrophages into the Infected Cochlea (A) Decalcified cochleas were cleared with an Organ Clearing kit. (B) The 3D structure of the right cochlea was imaged with a Lightsheet microscope to identify neutrophils (green) 1 day after in vivo P. aeruginosa infection of Coch+/+ LysM-GFP+/ and Coch/ LysM-GFP+/ mice. Clustered neutrophils (yellow arrowheads) were more abundant in the scala tympani of Coch+/+ than Coch/ mice. Single immune cells with GFP signal were rarely visualized in Coch/ mice (orange arrowheads). Instead, enlarged vessels in the spiral ligament (green-colored autofluorescence between two white lines in blue crop) were exclusively prominent in Coch/ mice, indicating the inflammatory stage in the scala media. t, scala tympani; m, scala media; v, scala vestibuli. (C) Recruited neutrophils in the 3D image of cochlea in wild-type versus Coch/ mice (n = 5–8 replicates from 2 mice in each group). ***p < 0.001. (D) Neutrophils and macrophages were counted in the Coch+/+ LysM-GFP+/ and Coch/ LysM-GFP+/ mice 1 day after in vivo P. aeruginosa infection by FACS. CD11b+/CD11c/Ly6G+ cells were identified as neutrophils (n = 6 mice in each group) and CD11b+/CD11c+/CD64+/F4/80+ cells were identified as macrophages (n = 5 mice in each group). *p < 0.05. (E) Representative whole-mount staining images of macrophage infiltration (F4/80, green) into the spiral ligament and spiral ganglion 1 day after in vivo P. aeruginosa infection. Staining highlights morphological differences between the resident (white arrowheads, magnified in the upper right inset) and infiltrated (yellow arrowheads, magnified in the upper right inset) macrophages. Red, cochlin; blue, nuclei/DAPI; green, F4/80. Scale bar, 40 mm. See also Figure S5 and Videos S1 and S2.

Figure 6E). Using scanning electron microscopy (SEM), we simi- for 7 days to generate chronic middle ear infection, Coch/ larly observed that the PAO1 bacteria were more aggregated af- mice showed more severe inflammation and residual bacteria ter LCCL supplementation and appeared to be morphologically in the middle ear, highly reminiscent of the state of chronic otitis entrapped into complex extracellular formations, namely beads media in human (Figure S6D). Therefore, cochlin is also effective and strings, (orange arrowheads in Figure 6F). Notably, these ef- on the prevention of outgrowth of P. aeruginosa causing chronic fects were exclusively observed after LCCL addition. otitis media, although a systemic immune response from the co- Next, we investigated the kinds of bacteria LCCL affected chlin in the spleen may be involved. and which part of the bacteria LCCL bound. An isolated We also examined the effect of LCCL on Staphylococcus P. aeruginosa from a patient with chronic otitis media for several aureus, representing gram-positive bacteria, and pathogens of decades (Figure S6A) was also aggregated and the growth was acute otitis media including Haemophilus influenzae and Strep- inhibited by LCCL (Figures S6B and S6C). Thus, LCCL also ex- tococcus pneumoniae. We observed that both S. aureus and erts an anti-bacterial effect on pathogenic P. aeruginosa. H. influenzae were aggregated in an LCCL-dependent manner Furthermore, when we inoculated GFP-PAO1 in the ear drum (Figure 7A). S. aureus infection in the cochlea dominantly

Cell Host & Microbe 25, 1–13, April 10, 2019 7 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

Figure 6. The Cleaved Cochlin LCCL Domain Directly Binds to and Aggregates P. aeruginosa Bacteria (A) Purified LCCL-His peptide was qualified with comparison to the LCCL blot in PAO1-infected cochlea lysate (left panel). For in vitro experiments with the LCCL domain, 1 mg/mL LCCL peptide was used. Bacterial growth curve in the presence and absence of LCCL (not significant, by repeat-measure ANOVA, n = 6 replicates in each group) (right panel). (B) To obtain the exact bacterial count by colony-forming unit (CFU) and compare the morphological difference of the colonies, a total of 103 CFUs of PAO1 were incubated at 37C for 12 h, serially diluted, and spread on LB agar plates with or without LCCL peptide (1 mg/mL). The pattern and number of grown colonies of GFP-PAO1 bacteria are shown (n = 5 replicates in each group). ns, not significant by paired t test. (C) When PAO1-GFP was cultured for 6 h on coverslips without shaking, multiple clusters of GFP-PAO1 were identified when treated with LCCL, whereas the bacteria were evenly grown as single bacillus or diplobacilli without LCCL. Scale bars, 20 mm. (D) The GFP signal of GFP-PAO1 alone or after LCCL treatment is shown (n = 9 replicates in each group). Aggregated GFP-PAO1 clusters were only observed after the addition of LCCL (n = 9 replicates in each group). **p < 0.01; ***p < 0.001 by paired t test. (E) Representative immunostaining images of PAO1-GFP (green) and LCCL (red) co-localization (white arrowheads). Scale bars, 20 mm. (F) Scanning electron microscopy images showing the growth pattern of GFP-PAO1 under non-shaking conditions with and without LCCL treatment. LCCL induced bacterial aggregation (white arrowheads) and entrapment (yellow arrowheads) into complex bead and string formations (red arrowheads). See also Figure S6 and Video S3. increased IL-1b and IL-6, as did P. aeruginosa (Figure S7A). In manner (Brown et al., 2018). Indeed, the globular aggregation addition, the growth of S. aureus and S. pneumoniae was in- of PAO1 by LCCL was partially dissociated following calcium hibited by cochlin in the cochlea (Figure S7B). From these depletion by ethylenediaminetetraacetic acid (EDTA; Figure 7B; data, we consider that the effect of LCCL is independent of Video S4). This implied that LCCL-induced bacterial aggrega- LPS because gram-positive bacteria such as S. aureus and tion is calcium dependent. Next, we performed biofilm assays S. pneumoniae were also affected by LCCL. Consistent with to determine whether the interaction promotes biofilm forma- this, a lipid A mutant (HtrB1 [PA0011], acyltransferase mutant) tion. However, there was no difference in P. aeruginosa biofilm of PAO1 showed the same aggregation as PAO1 upon LCCL formation regardless of LCCL (Figures 7C and 7D). We then addition (Figure 7A). conducted a transwell assay to investigate whether LCCL Finally, we examined how the interaction between LCCL and directly plays a role as a chemoattractant on neutrophil migra- bacteria modulates the innate immune response. As C-type tion. Neutrophil number in the lower chamber in the presence of lectin, functioning as an opsin that facilitates antimicrobial im- LCCL was significantly higher compared to that of the control. munity, has calcium-dependent activity, we reasoned that the Thus, LCCL has a direct chemotactic role to induce neutrophil LCCL domain may aggregate bacteria in a calcium-dependent migration (Figure 7E). To further confirm the direct binding

8 Cell Host & Microbe 25, 1–13, April 10, 2019 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

Figure 7. LCCL Domain Aggregates Bacteria Independently of LPS and Directly Chemoattracts Neutrophils (A) Gram-positive S. aureus (GFP-tagged) and gram-negative H. influenzae were prepared. As a lipid A mutant, acyltransferase HtrB1 P. aeruginosa mutant (PA0011) was generated from the PAO1 strain. In the presence of the LCCL domain (6 h incubation), S. aureus, H. influenzae, and PA0011 (green, pseudo color) were more aggregated compared to the control with culture media (white arrowheads). Scale bars, 20 mm. (B) Aggregation of P. aeruginosa by LCCL is dependent on calcium. GFP-PAO1 globular aggregation by LCCL (1 mg/mL) was observed on the coverslip and partially dissociated following calcium depletion by ethylenediaminetetraacetic acid (EDTA, 10 mM in pH 7.4). Scale bar, 20 mm. (C) The extent of P. aeruginosa (PAO1) biofilm formation was measured using the dye crystal violet (left panel). Biofilm formation of PAO1 was quantified by microtiter plate assay according to the presence of LCCL peptide (1 mg/mL) (n = 10 replicates in each group, right panel). ns, not significant by paired t test. (D) In the scanning electron microscopy image, biofilms of P. aeruginosa cultured on the coverslip in 6-well plate were identified with or without LCCL peptide (1 mg/mL). There was no morphological difference between the two groups. (E) Transwell assay depending on the presence or absence of LCCL (1 mg of LCCL peptide in the lower chamber). Isolated neutrophils from mouse femur and tibia bone marrow were added into Matrigel. After 2 h incubation, migrated neutrophils in the lower chamber were counted in the field of view (FOV, n = 5 replicates in each group). ***p < 0.001 by Student’s t test. See also Figure S7 and Video S4.

between neutrophils and LCCL, we investigated the binding af- DISCUSSION finity using microscopy. Although confocal microscopy for anti- LCCL antibody labeling of neutrophils revealed a weak affinity The cochlea is a specialized organ that is differentiated into to LCCL (data not shown), SEM verified that extracellular en- various compartments. The organ of Corti, located in the scala tities were bound to the LCCL-treated neutrophil surface (Fig- media, is surrounded by endolymph and has relatively limited im- ure S7C). From these data, we speculate that the entities on mune capacity because the cochlea is isolated from the sys- the neutrophil surface may be LCCL itself or at least generated temic immune system by the blood-labyrinthine barrier (Du from the interaction between LCCL and neutrophils. Overall, et al., 2011; Jung et al., 2015; Robertson et al., 2008). Although LCCL appears to directly interact with bacteria, thus mediating this barrier limits systemic immune cell infiltration, there are their segregation and entrapment, eventually leading to a local- many resident immune-related cells found in the lateral wall ized chemoattraction of immune cells and an enriched innate and basilar membrane, which are activated by acoustic injury immune response in the inner ear while protecting essential or labyrinthitis (Hu et al., 2018; Vethanayagam et al., 2016). auditory structures. These consist of supporting cells, fibrocytes, macrophages,

Cell Host & Microbe 25, 1–13, April 10, 2019 9 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

and dendritic cells that can differentiate into antigen-presenting indicating that this factor and/or its cleaved LCCL domain cells. Thus, the innate immune response in the spiral ligament, can also affect the immune response in peripheral tissues. spiral limbus, neural tissue, basilar membrane, and other com- The expression of cochlin in the inner ear and eye can, there- partments, such as the scala tympani and vestibule, is critical fore, be largely explained by their separation from the systemic to prevent sensory epithelial organ damage. However, the immune response, which likely relies on cochlin expression in spatiotemporal control of the innate immune response, which the spleen. eliminates pathogens while protecting essential auditory struc- The mechanism of cochlin is highlighted by our ex vivo exper- tures, is not well understood. In this study, we evaluated the imental data, which show that cochlea-derived cochlin suffi- role of cochlin in this process during bacterial infection of the in- ciently modulates the innate immune response within the same ner ear. Our data indicated that cochlin in the inner ear acts as a tissue via secretion of its cleaved LCCL domain. This domain surveillance system against bacterial infection in the perilymph. has an anti-bacterial effect and inhibits bacterial overgrowth Upon infection, cochlin is cleaved and the LCCL-domain-con- within the intra-organ milieu by sufficiently promoting resident taining fragment is secreted into the scala tympani to form a de- immune cell infiltration in the cochlea along with aggregating fense network via pathogen segregation. This process enables and isolating the bacteria in the scala tympani. Notably, we the recruited neutrophils and monocytes to detect and eliminate demonstrate here that the LCCL domain directly interacts with the bacteria in addition to mediating a localized inflammatory the invading bacteria, whereas the current literature is conflicting cytokine response while restraining the access of pathogens, as to how LCCL modulates its effects. It was previously reported as well as that of immune cells, to the organ of Corti. In summary, that the cleaved cochlin LCCL domain did not directly affect bac- cochlin, via LCCL domain cleavage and secretion, plays a signif- terial viability or growth in vitro nor have a direct effect on macro- icant role in preserving critical sensory organ structure and audi- phage activation or cytokine secretion (Py et al., 2013). However, tory function. a recent study showed that the LCCL domain increased tyrosine Although innate immune response is important to maintain phosphorylation of murine macrophages, indicating cellular acti- auditory organ function, an excessive immune reaction can vation (Nystro¨ m et al., 2018). deteriorate the organ of Corti (Tornabene et al., 2006; Woo In the present study, we found that cochlin, specifically the et al., 2015). In fact, the activation of innate immunity in the cleaved LCCL domain, directly binds to P. aeruginosa, leading cochlea following pathogenic infections and acoustic trauma to agglutination, which differs from the concept of conventional often induces an excessive inflammatory response, leading biofilm as shown in Figures 7C and 7D. It is still unclear where to aberrant hearing deterioration (Fujioka et al., 2006; Hirose the LCCL domain binds among the bacterial compartments. et al., 2005; Tornabene et al., 2006). Abolishing the expression The literature is inconclusive regarding whether LCCL domain of TLR4 or pro-inflammatory cytokines, such as IL-1b and binds to LPS or its component, lipid A (Liepinsh et al., 2001; Py IL-6, appears to ameliorate sensory organ deterioration after et al., 2013; Va´ sa´ rhelyi et al., 2014). The present study suggests acoustic trauma (Fujioka et al., 2006; Jones et al., 2011; Ve- that LPS is not the necessary factor for LCCL domain-bacteria thanayagam et al., 2016) but may also limit pathogen elimina- interaction, because this domain also has the same effect on tion. Furthermore, anti-inflammatory cytokines, such as IL-10, lipid A mutant P. aeruginosa and on both gram-positive and also alter inflammatory pathways in the cochlea and inhibit nu- gram-negative bacteria. However, it is still possible that it may clear factor kappa-light-chain-enhancer of activated B cells directly interact with adaptor proteins that are relevant to LPS, (NF-kB) activation (Woo et al., 2015). Therefore, balanced given that lipid A has been reported to bind to the LCCL domains coordination between these pro- and anti-inflammatory re- of CRISPLD2 (Lgl1), which subsequently recruits immune cells to sponses is essential to protect the organs of the inner ear the bound and segregated pathogens in the lung (Va´ sa´ rhelyi from pathogens and reduce post-inflammatory tissue dam- et al., 2014). Moreover, in our SEM and live-imaging analyses, age. In this regard, cochlin appears to narrow the inflamma- we also observed the LCCL domain to affect the bacterial sur- tory field down to a limited space to preserve the organ of face membrane, possibly by altering hydrophobicity or inducing Corti. This mechanism is important for cochlear homeostasis cytoskeleton changes. Thus, our evidence strongly supports and function because immune cell migration to the sensory direct binding of the cochlin LCCL domain to pathogenic hair cells may increase collateral damage in the organ of Corti. bacteria. Cochin increases the efficacy of pathogen elimination while Another mechanism of cochlin is a direct chemotactic effect of also reducing organ damage owing to the overstimulated in- neutrophils. LCCL clearly increased neutrophil recruitment by flammatory reaction. around 10-fold (Figure 7E). With regard to the role of LCCL as The immune response mediated by the LCCL-domain-con- a chemoattractant, the direction of LCCL secretion in the co- taining fragment of cochlin is limited by many immune barriers chlea is notable. Because LCCL is mainly secreted into the scala and specialized fluid-filled compartments within the cochlea tympani, the attracted immune cells tend to induce innate inflam- that hinder immune cell infiltration and efficient pathogen mation only in the perilymph space but not the organ of Corti. The detection. Thus, local cochlin expression within the cochlea trajectory of the conduits for LCCL secretion is indispensable in is evolutionarily advantageous, as it mediates a local the cochlea for the prevention of sensory organ and hearing response. However, cochlea-derived cochlin, therefore, likely damage. It will be promising to develop anti-bacterial therapeu- only affects the local environment in the inner ear. This is in tics using LCCL peptide in patients with chronic otitis media slight contrast to a previous study in which cochlin from given that LCCL has a direct chemotaxis effect, validated against the spleen could systemically modulate an innate immune patient-derived P. aeruginosa, for the enhancement of immune response in the lung after bacterial infection (Py et al., 2013), response.

10 Cell Host & Microbe 25, 1–13, April 10, 2019 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

The important function of cochlin in the inner ear was previ- of LCCL domain-containing peptide fragments rescued hear- ously foreshadowed by the clinical manifestation of COCH ing deterioration after bacterial infection, this domain may be mutations as inherited progressive sensorineural hearing a potential therapeutic drug candidate to prevent sensori- loss (DFNA9). Cochlin LCCL domain cleavage is significantly neural hearing loss associated with chronic otitis media. decreased in the COCH mutants, implying that the uncleaved Although additional work is necessary, this study highlights LCCL domain does not mediate an inflammatory response in a distinct role for cochlin in pathogen surveillance in the the inner ear (Jung et al., 2015). Although DFNA9 pathogenesis inner ear. is not completely clear, a dominant-negative effect is thought to induce the DFNA9 phenotype, particularly as hearing in Coch STAR+METHODS null mice is not defective (Jones et al., 2011; Makishima et al., 2005). Indeed, absence of LCCL in the cochlea alone Detailed methods are provided in the online version of this paper does not appear to be related to DFNA9 pathology. However, and include the following: heterologous p.G88E knockin mice mimic the DFNA9 phenotype (Robertson et al., 2008), implying that aggregation of mis- d KEY RESOURCES TABLE folded and uncleaved cochlin, also termed an accumulated d CONTACT FOR REAGENT AND RESOURCE SHARING acidophilic substance, in the inner ear may have cytotoxic ef- d EXPERIMENTAL MODEL AND SUBJECT DETAILS fects on the cochlea organ in patients with DFNA9. Neverthe- B Human Subjects less, the possibility that the immunocompromised condition B Animals and Cochlea Explants of Coch/ mice or humans may aggravate hearing loss over B Cell Lines and Heterologous Transfection time and increase the chance of pathogen invasion or acoustic d METHOD DETAILS trauma cannot be ruled out. This phenomenon is difficult to B P. aeruginosa Infection Model study in mice owing to the specific pathogen-free conditions B Quantitative Real-time PCR in which they are maintained. As most studies reporting cases B Western Blotting Analysis of DFNA9 utilized criteria that excluded patients that had been B Immunostaining and Whole-mount ImmunoFluo- treated for an infectious disease in the ear, it is necessary to rescence investigate whether these patients have single nucleotide poly- B In situ Hybridization morphisms or mutations in COCH that could increase their sus- B Flow Cytometry ceptibility to inner ear infection. B Enzyme-Linked Immunosorbent Assay Glaucoma has also been linked to cochlin, although the B Mouse Neutrophil Isolation mechanism remains elusive (Bhattacharya et al., 2005a; Rob- B Scanning Electron Microscopy ertson et al., 1998, 2008). The inner ear and the eye are similar B Tissue Clearing and Immunostaining in terms of the expression of certain proteins including LCCL- B Single Plane Illumination Microscopy containing molecules such as cochlin and vitrin (akhirin) (Ahsan B Auditory Function Test et al., 2005; Bishop et al., 2002). This may reflect common roles B Biofilm Assay for these proteins in the aqueous spaces of these two sensory B Transwell Assay organs, although there is no evidence that patients with muta- B Liquid Chromatography-Tandem Mass Spectrometry tions in COCH or VIT are more susceptible to bacterial infection d QUANTIFICATION AND STATISTICAL ANALYSIS in the inner ear or the eye. In fact, the function of the LCCL d DATA AND SOFTWARE AVAILABILITY domain in these proteins is quite similar to that in soluble C-type lectin, which facilitates phagocytic uptake, activates SUPPLEMENTAL INFORMATION the complement pathway, and modulates the direct killing of pathogens (Brown et al., 2018). However, the LCCL domain Supplemental Information can be found with this article online at https://doi. org/10.1016/j.chom.2019.02.001. in this protein has neither EPN (Glu-Pro-Asn) nor QPD (Gln-Pro-Asp) motifs, which are responsible for carbohydrate ACKNOWLEDGMENTS binding. This LCCL domain also does not bind to glucose, mannose, cellobiose, or 2-N-acetyl-glucosamine (Liepinsh This work was supported by National Research Foundation of Korea (NRF) et al., 2001). Therefore, it is unlikely that the LCCL domain is grants funded by the Korean government (2017R1D1A1B03030046 a canonical C-type lectin protein. Given that this domain facil- and 2017M3A9E8029721 to J.J., 2014M3A9D5A01073865 to J.Y.C., and itates innate immune activity by direct interaction with and ag- 2016R1A2B4008199 to Y.-M.H.). gregation of pathogens in a calcium-dependent manner, it may be a member of the broad category of C-type lectin-like do- AUTHOR CONTRIBUTIONS mains. Further biochemical study is necessary to fully charac- Study Conception and Design, J.J., Y.-M.H., and J.Y.C.; Acquisition of Data, terize the LCCL domain. J.J., J.E.Y., Y.H.C., and G.-Y.K.; Analysis and Interpretation of Data, J.J., In conclusion, we investigated the role of cochlin in the inner J.E.Y., Y.H.C., and G.-Y.K.; Drafting of Manuscript, J.J., Y.-M.H., and J.Y.C.; ear during bacterial infection. Our data indicate that cleaved Technical Support, D.S.J., S.S.Y., and K.-M.L.; Essential Consulting and Re- cochlin LCCL in the cochlea has a critical role in enhancing agents, S.C.P., Hack June Lee, Hyun Jae Lee, B.N., S.H.K., and J.-H.Y. the innate immune response by conglomerating the pathogens DECLARATION OF INTERESTS within a limited space and recruiting local innate immune cells and cytokines directly to their position. Given that the addition The authors declare no competing interests.

Cell Host & Microbe 25, 1–13, April 10, 2019 11 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

Received: August 15, 2018 Jung, J., Kim, H.S., Lee, M.G., Yang, E.J., and Choi, J.Y. (2015). Novel COCH Revised: December 14, 2018 p.V123E mutation, causative of DFNA9 sensorineural hearing loss and vestib- Accepted: February 5, 2019 ular disorder, shows impaired Cochlin post-translational cleavage and secre- Published: March 21, 2019 tion. Hum. Mutat. 36, 1168–1175. Jung, J., Kim, J., Roh, S.H., Jun, I., Sampson, R.D., Gee, H.Y., Choi, J.Y., REFERENCES and Lee, M.G. (2016). The HSP70 co-chaperone DNAJC14 targets mis- folded pendrin for unconventional protein secretion. Nat. Commun. Ahsan, M., Ohta, K., Kuriyama, S., and Tanaka, H. (2005). Novel soluble mole- 7, 11386. cule, Akhirin, is expressed in the embryonic chick eyes and exhibits hetero- philic cell-adhesion activity. Dev. Dyn. 233, 95–104. Kawauchi, H., DeMaria, T.F., and Lim, D.J. (1989). Endotoxin permeability through the round window. Acta Otolaryngol. Suppl. 457, 100–115. Bahk, Y.Y., Kim, S.A., Kim, J.S., Euh, H.J., Bai, G.H., Cho, S.N., and Kim, Y.S. (2004). Antigens secreted from Mycobacterium tuberculosis: identification Lee, K.M., Go, J., Yoon, M.Y., Park, Y., Kim, S.C., Yong, D.E., and Yoon, S.S. by proteomics approach and test for diagnostic marker. Proteomics 4, (2012). Vitamin B12-mediated restoration of defective anaerobic growth leads 3299–3307. to reduced biofilm formation in Pseudomonas aeruginosa. Infect. Immun. 80, Bhattacharya, S.K., Peachey, N.S., and Crabb, J.W. (2005a). Cochlin and glau- 1639–1649. coma: a mini-review. Vis. Neurosci. 22, 605–613. Liepinsh, E., Trexler, M., Kaikkonen, A., Weigelt, J., Ba´ nyai, L., Patthy, L., and Bhattacharya, S.K., Rockwood, E.J., Smith, S.D., Bonilha, V.L., Crabb, J.S., Otting, G. (2001). NMR structure of the LCCL domain and implications for Kuchtey, R.W., Robertson, N.G., Peachey, N.S., Morton, C.C., and Crabb, DFNA9 deafness disorder. EMBO J. 20, 5347–5353. J.W. (2005b). Proteomics reveal Cochlin deposits associated with glaucoma- Makishima, T., Rodriguez, C.I., Robertson, N.G., Morton, C.C., Stewart, C.L., tous trabecular meshwork. J. Biol. Chem. 280, 6080–6084. and Griffith, A.J. (2005). Targeted disruption of mouse Coch provides func- Bishop, P.N., Takanosu, M., Le Goff, M., and Mayne, R. (2002). The role of the tional evidence that DFNA9 hearing loss is not a COCH haploinsufficiency dis- posterior ciliary body in the biosynthesis of vitreous humour. Eye 16, 454–460. order. Hum. Genet. 118, 29–34. Brown, G.D., Willment, J.A., and Whitehead, L. (2018). C-type lectins in immu- Mittal, R., Lisi, C.V., Gerring, R., Mittal, J., Mathee, K., Narasimhan, G., Azad, nity and homeostasis. Nat. Rev. Immunol. 18, 374–389. R.K., Yao, Q., Grati, M., Yan, D., et al. (2015). Current concepts in the patho- Cai, Q., Vethanayagam, R.R., Yang, S., Bard, J., Jamison, J., Cartwright, D., genesis and treatment of chronic suppurative otitis media. J. Med. Dong, Y., and Hu, B.H. (2014). Molecular profile of cochlear immunity in the Microbiol. 64, 1103–1116. resident cells of the organ of Corti. J. Neuroinflamm. 11, 173. Moon, S.K., Park, R., Lee, H.Y., Nam, G.J., Cha, K., Andalibi, A., and Lim, D.J. Chung, K., Wallace, J., Kim, S.Y., Kalyanasundaram, S., Andalman, A.S., (2006). Spiral ligament fibrocytes release chemokines in response to otitis me- Davidson, T.J., Mirzabekov, J.J., Zalocusky, K.A., Mattis, J., Denisin, A.K., dia pathogens. Acta Otolaryngol. 126, 564–569. et al. (2013). Structural and molecular interrogation of intact biological sys- Muta, T., Miyata, T., Misumi, Y., Tokunaga, F., Nakamura, T., Toh, Y., tems. Nature 497, 332–337. Ikehara, Y., and Iwanaga, S. (1991). Limulus factor C. An endotoxin-sensi- Du, X., Choi, C.H., Chen, K., Cheng, W., Floyd, R.A., and Kopke, R.D. tive serine protease zymogen with a mosaic structure of complement-like, (2011). Reduced formation of oxidative stress biomarkers and epidermal growth factor-like, and lectin-like domains. J. Biol. Chem. 266, migration of mononuclear phagocytes in the cochleae of chinchilla after 6554–6561. antioxidant treatment in acute acoustic trauma. Int. J. Otolaryngol. Nystro¨ m, A., Bornert, O., Kuhl,€ T., Gretzmeier, C., Thriene, K., Dengjel, J., 2011, 612690. Pfister-Wartha, A., Kiritsi, D., and Bruckner-Tuderman, L. (2018). Impaired Engel, F., Blatz, R., Kellner, J., Palmer, M., Weller, U., and Bhadki, S. (1995). lymphoid extracellular matrix impedes antibacterial immunity in epidermolysis Breakdown of the round window membrane permeability barrier evoked by bullosa. Proc. Natl. Acad. Sci. USA 115, E705–E714. streptolysin O: possible etiologic role in development of sensorineural hearing O’Toole, G.A. (2011). Microtiter dish biofilm formation assay. J. Vis. Exp. 47. loss in acute otitis media. Infect. Immun. 63, 1305–1310. Py, B.F., Gonzalez, S.F., Long, K., Kim, M.S., Kim, Y.A., Zhu, H., Yao, J.H., Faust,N.,Varas,F.,Kelly,L.M.,Heck,S.,andGraf,T.(2000).Insertion Degauque, N., Villet, R., Ymele-Leki, P., et al. (2013). Cochlin produced by of enhanced green fluorescent protein into the lysozyme gene creates follicular dendritic cells promotes antibacterial innate immunity. Immunity 38, mice with green fluorescent granulocytes and macrophages. Blood 96, 1063–1072. 719–726. Fujioka, M., Kanzaki, S., Okano, H.J., Masuda, M., Ogawa, K., and Okano, H. Robertson, N.G., Khetarpal, U., Gutie´ rrez-Espeleta, G.A., Bieber, F.R., and (2006). Proinflammatory cytokines expression in noise-induced damaged co- Morton, C.C. (1994). Isolation of novel and known genes from a human fetal chlea. J. Neurosci. Res. 83, 575–583. cochlear cDNA library using subtractive hybridization and differential screening. Genomics 23, 42–50. Hirose, K., Discolo, C.M., Keasler, J.R., and Ransohoff, R. (2005). Mononuclear phagocytes migrate into the murine cochlea after acoustic Robertson, N.G., Lu, L., Heller, S., Merchant, S.N., Eavey, R.D., McKenna, trauma. J. Comp. Neurol. 489, 180–194. M., Nadol, J.B., Jr., Miyamoto, R.T., Linthicum, F.H., Jr., Lubianca Neto, Hu, B.H., Zhang, C., and Frye, M.D. (2018). Immune cells and non-im- J.F., et al. (1998). Mutations in a novel cochlear gene cause DFNA9, a hu- mune cells with immune function in mammalian cochleae. Hear. Res. man nonsyndromic deafness with vestibular dysfunction. Nat. Genet. 20, 362, 14–24. 299–303. Ikezono, T., Shindo, S., Li, L., Omori, A., Ichinose, S., Watanabe, A., Robertson, N.G., Jones, S.M., Sivakumaran, T.A., Giersch, A.B., Jurado, S.A., Kobayashi, T., Pawankar, R., and Yagi, T. (2004). Identification of a novel Call, L.M., Miller, C.E., Maison, S.F., Liberman, M.C., and Morton, C.C. (2008). Cochlin isoform in the perilymph: insights to Cochlin function and the A targeted Coch missense mutation: a knock-in mouse model for DFNA9 late- pathogenesis of DFNA9. Biochem. Biophys. Res. Commun. 314, onset hearing loss and vestibular dysfunction. Hum. Mol. Genet. 17, 440–446. 3426–3434. Jones, S.M., Robertson, N.G., Given, S., Giersch, A.B., Liberman, M.C., and Ryu, J.C., Kim, M.J., Kwon, Y., Oh, J.H., Yoon, S.S., Shin, S.J., Yoon, J.H., and Morton, C.C. (2011). Hearing and vestibular deficits in the Coch(-/-) null mouse Ryu, J.H. (2017). Neutrophil pyroptosis mediates pathology of P. aeruginosa model: comparison to the Coch(G88E/G88E) mouse and to DFNA9 hearing lung infection in the absence of the NADPH oxidase NOX2. Mucosal and balance disorder. Hear. Res. 272, 42–48. Immunol. 10, 757–774. Jung, J., Nam, J.H., Park, H.W., Oh, U., Yoon, J.H., and Lee, M.G. (2013). Tornabene, S.V., Sato, K., Pham, L., Billings, P., and Keithley, E.M. (2006). Dynamic modulation of ANO1/TMEM16A HCO3(-) permeability by Ca2+/ Immune cell recruitment following acoustic trauma. Hear. Res. 222, calmodulin. Proc. Natl. Acad. Sci. USA 110, 360–365. 115–124.

12 Cell Host & Microbe 25, 1–13, April 10, 2019 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

Va´ sa´ rhelyi, V., Trexler, M., and Patthy, L. (2014). Both LCCL-domains of hu- Woo, J.I., Kil, S.H., Oh, S., Lee, Y.J., Park, R., Lim, D.J., and Moon, S.K. (2015). man CRISPLD2 have high affinity for lipid A. Biochimie 97, 66–71. IL-10/HMOX1 signaling modulates cochlear inflammation via negative regulation of MCP-1/CCL2 expression in cochlear fibrocytes. J. Immunol. 194, Vethanayagam, R.R., Yang, W., Dong, Y., and Hu, B.H. (2016). Toll-like recep- 3953–3961. tor 4 modulates the cochlear immune response to acoustic injury. Cell Death Zuo, X., Echan, L., Hembach, P., Tang, H.Y., Speicher, K.D., Santoli, D., and Dis. 7, e2245. Speicher, D.W. (2001). Towards global analysis of mammalian proteomes us- Wilkinson, D.G., Bailes, J.A., and McMahon, A.P. (1987). Expression of the ing sample prefractionation prior to narrow pH range two-dimensional gels and proto-oncogene int-1 is restricted to specific neural cells in the developing using one-dimensional gels for insoluble and large proteins. Electrophoresis mouse embryo. Cell 50, 79–88. 22, 1603–1615.

Cell Host & Microbe 25, 1–13, April 10, 2019 13 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Cochlin Millipore Cat# 9A10D2 Anti-Cochlin NOVUS Cat# NBP1-69141; RRID: AB_11038442 Anti-Cochlin Santa Cruz Cat# sc-67220; RRID: AB_2083669 Anti-Myosin 7a Santa Cruz Cat# sc-74516; RRID: AB_2148626 Anti-b-actin Santa Cruz Cat# sc-47778; RRID: AB_2714189 Anti-IL 1b Abcam Cat# ab9722; RRID: AB_308765 Anti-F4/80 Abcam Cat# ab6640; RRID: AB_1140040 Anti-I-A/I-E BioLegend Cat# 107601; RRID: AB_313316 Anti-CD64 PE BioLegend Cat# 139304; RRID: AB_10612740 Anti-F4/80 APC eBioscience Cat# 17-4801-82; RRID: AB_2735035 Anti-CD115 LSBio Cat# LS-C164350 Anti-CD11c PE-Cy7 BD Bioscience Cat# 558079; RRID: AB_647251 Anti-CD11b FITC BD Bioscience Cat# 553310; RRID: AB_394774 Anti-Ly6G APC Biolegend Cat# 552126; RRID: AB_1877212 Anti-F4/80 PE BD Bioscience Cat# 565410; RRID: AB_2687527 Anti-Ly6G BioLegend Cat# 127608; RRID: AB_1186099 Anti-Ly6C BD Bioscience Cat# 560595; RRID: AB_1727554 Anti-myc Cell Signaling Cat# 2276; RRID: AB_331783 Bacterial and Virus Strains Pseudomonas aeruginosa ATCC Cat# 15692 Staphylococcus aureus ATCC Cat# 29213 Streptococcus pneumoniae ATCC Cat# 15898 Haemophilus inﬂuenzae ATCC Cat# 49766 (from Korean Culture Center for Microorganisms) Biological Samples Ear discharge from the patient with chronic otitis media This paper N/A Chemicals, Peptides, and Recombinant Proteins LPS Sigma-Aldrich Cat# L2630 MMP inhibitor lll Santa Cruz Cat# SC-311427 Human LCCL peptide (short isoform cochlin) Sino Biological Cat# 11368-H07HS PNGase F BioLabs Cat# P0704S APC conjugation Kit Abcam Cat# Ab201807 Mounting solution Sigma-Aldrich Cat# M7534 ELISA substrate reagent pack R&D systems Cat# DY999 Critical Commercial Assays ELISA kit: Mouse IL-1b R&D Systems Cat# DY401 ELISA kit: Mouse IL-6 R&D Systems Cat# DY406 ELISA kit: Mouse CCL2/MCP-1 R&D Systems Cat# DY479-05 ELISA kit: Mouse CXCL1/KC R&D Systems Cat# DY453-05 ELISA kit: Mouse CXCL2/MIP-2 R&D Systems Cat# MM200 Lipofectamine Plus Reagent ThermoFischer Cat# 15338100 jetPRIME reagent Polyplus Cat# 114-07 qPCR Master Mix (2X) KAPA Biosystems Cat# KR0390 (Continued on next page)

e1 Cell Host & Microbe 25, 1–13.e1–e6, April 10, 2019 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

Continued REAGENT or RESOURCE SOURCE IDENTIFIER TaqMan Universal Master Mix ll ThermoFischer Cat# 4440038 iScript cDNA Synthesis Kit Biorad Cat# 1708891 EasySepTM mouse neutrophil enrichment kit Stemcell technologies Cat# 19762 Experimental Models: Cell Lines HEK293 CLS Cat# 300192/p777_HEK293; RRID: CVCL_0045 Experimental Models: Organisms/Strains B6.129S1(Cg)-Cochtm1.1Stw/YuanJ The Jackson Laboratory Cat# 024691 GFP-LysM+/ Faust et al. (2000) N/A Oligonucleotides siRNA targeting sequence: Human ADAMTS4 Dharmacon Cat# L-003807-00-0005 siRNA targeting sequence: Human ADAMTS5 Dharmacon Cat# E-005775-00-0005 Primers for qPCR, see Table S1 This paper N/A Primers for PAO1 deletion, see Table S1 This paper N/A Primers for in situ hybridization, see Table S1 This paper N/A Recombinant DNA pCMV3-human COCH-myc Sino Biological Cat# HG11368-CM Software and Algorithms Flowjo FLOWJO, LLC https://www.flowjo.com/ Prism GraphPad https://www.graphpad.com/ scientificsoftware/prism/ SPSS v.21 SPSS https://www.ibm.com/analytics/spss- statistics-software/ Arivis Vision 4D Arivis https://www.arivis.com/en/imaging- science/arivis-vision4d Imaris Bitplane http://www.bitplane.com/ Turbo Sequest ThermoFischer https://www.thermofisher.com/ MASCOT program Matrix Science http://www.matrixscience.com

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact, Jae Young Choi ([email protected]).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Human Subjects For the case analysis and isolation of P. aeruginosa from the ear with chronic otitis media, the study was approved by the Institutional Review Board of the Severance Hospital, Yonsei University Health System (IRB#4-2018-0577). One subject was enrolled. She was 58 years-old female and suffered from chronic otitis media combined with sensorineural hearing loss. P. aeruginosa was isolated from the bacterial culture of the patient’s ear discharge.

Animals and Cochlea Explants Coch/ knockout mice on a C57B6 background were commercially purchased from Jackson Laboratory (Strain name: B6.129S1(Cg)-Cochtm1.1Stw/YuanJ). Mice with enhanced green ﬂuorescent protein inserted into the lysozyme M gene (LysM-GFP) (Faust et al., 2000) were also maintained, and heterogenetic mice (GFP+/-) were used in our experiments. All mice were embryo-transferred and housed under speciﬁc pathogen-free conditions at the animal facility of Yonsei University. The animal ethics committee of Yonsei University College of Medicine approved all of the experimental protocols used (2015-0127). All experiments with live mice were performed under general anesthesia with tiletamine mixed with zolazepam (100 mg/kg) and xylazine (50 mg/kg) via intraperi- toneal injection. For P1–3 mice, cold ice anesthesia was applied in some cases. For ex vivo culture of the cochlea, the cochlea was dissected from 4-week-old male mice and placed in a culture plate. After removing the temporal bone and placing the cochlea in phosphate buffered saline (PBS; pH 7.2), the soft connective tissues as well as the semi-circular canals and vestibules were removed.

Cell Host & Microbe 25, 1–13.e1–e6, April 10, 2019 e2 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

The cochlea explant was then placed on a culture plate and cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM)-based culture medium. All of the ex vivo experiments were performed immediately after explant preparation.

Cell Lines and Heterologous Transfection HEK 293 cell line (female origin) was maintained in high-glucose DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and penicillin (50 IU/mL)/streptomycin (50 mg/mL). The c-terminal myc-tagged expression vector for the coding region of human cochlin (pCMV3-hCOCH) was purchased from Sino Bio (Beijing, China). For transient transfection of the hCOCH construct into HEK293 cells, Lipofectamine Plus Reagent (Invitrogen) was used. For knockdown of aggrecanase 1 and 2, siRNA of each gene (ADAMTS4 and ADAMTS5) was purchased (Dharmacon; On-targetplus SMARTpool reagent) and transiently trans- fected into HEK 293 cells using jetPRIME reagent (Polyplus). P. aeruginosa Culture Conditions Preparation of P. aeruginosa PAO1 strain was performed as described previously (Ryu et al., 2017). Brieﬂy, PAO1 colonies on grown on a Luria-Bertani (LB) agar plate were inoculated into 10 mL of LB medium and grown in ambient atmospheric air at 37C overnight.

Samples of the overnight cultures were then grown with aeration until reaching an optical density (A600) of 1. The GFP-tagged PAO1 strain was kindly provided by Professor Jee-Hwan Ryu at Yonsei University and grown in LB supplemented with carbenicillin

(100 mg/mL). To obtain the growth curve of PAO1 with or without LCCL, the optical density (A600) was measured every hour up to 12 h. To obtain the exact bacterial count by colony forming unit (CFU) and compare the morphological difference of the colonies, a total of 103 CFUs of PAO1 were incubated at 37C for 12 h, serially diluted, then spread on the LB agar plate with or without LCCL peptide (1 mg/mL).

METHOD DETAILS

P. aeruginosa Infection Model P. aeruginosa is the most common pathogen that affects the chronic otitis media, and infection is known to cause hearing loss (Mittal et al., 2015). Thus, we chose this strain as the pathogenic bacterium for inner ear infection. To induce inner ear infection with PAO1, a total of 103 CFUs of live PAO1 suspended in 5 mL of LB medium were transtympanically inoculated into the posterior portion of middle ears (near the round window) of 4-week-old mice using a 30-G needle as described previously (Woo et al., 2015). As a control, LB media inoculation was performed in an independent group of mice. In the case of LCCL application, LCCL peptide (2.5 mLof 100 mg/mL) was applied transtympanically into the posterior protion of middle ears at the same time or 6 h later with P. aeruginosa inoculation (105 CFUs of live PAO1 suspended in 2.5 mL of LB medium). Mice were sacrificed 1 or 3 days after inoculation. For the ex vivo experiments, 107 CFUs of PAO1 were added to the explanted cochlea, whereas LB medium was applied into the control explanted cochlea. Other bacteria such as S. aureus and S. pneumoniae (from Professor Jee-Hwan Ryu) were incubated and prepared in a similar manner as P. aeruginosa. H. influenzae was obtained from Korean Culture Center for Microorganisms and cultured in chocolate agar plate and broth including dried bovine hemoglobin and Iso VitaleX. For lipid A mutant P. aeruginosa experiments, construction of a PA0011 (acyltransferase HtrB1) deletion mutant was conducted by allelic replacement as described previously (Lee et al., 2012). Briefly, approximately 600 bp of flanking sequences at both ends of each gene were amplified by PCR with the indicated primers. The upstream reverse primers and the downstream forward primers were complementary to each other. Thus, the 30 end of the upstream sequence and the 50 end of the downstream sequence were annealed together during PCR amplification. The deletion of PA0011 was confirmed by PCR and DNA sequencing.

Quantitative Real-time PCR qPCR was performed as described previously (Jung et al., 2013). To quantify mRNA of TLR4 as well as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 4 and ADAMTS 5 in the cochlea, gene-specific primer pairs with hsp80 control primer pairs were used in conjunction with a TaqMan probe (Applied Biosystems, Foster City, CA, USA). In addition, appropriate PCR primer pairs for defb3, defb4, reg3g, s100a8, and gapdh were achieved. To validate siRNAs of ADAMTS 4 and ADAMTS 5 in HEK 293 cells, PCR primer pairs for ADAMTS4, ADAMTS5, and GAPDH were also achieved. Purified RNA samples from the cochlea were reverse- transcribed using an iScript Select cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Amplification was performed using an ABI 7500 real-time PCR system (Applied Biosystems).

Western Blotting Analysis Western blotting was performed as described previously (Jung et al., 2016). For tissue western blotting, cochleae were frozen in liquid nitrogen, ground, and lysed in a sodium dodecyl sulfate (SDS) lysis buffer. For perilymph sampling, perilymph was collected by glass capillary aspiration from the round window of explanted cochlea. Lysed samples were mixed with sample buffer and separated by SDS-polyacrylamide gel electrophoresis. For digestion of glycosylated pendrin by N-Glycosidase F (PNGase F; New England Bio- labs), protein samples were incubated with PNGase F in solutions containing triton X-100 (1%) for 2 h. The separated proteins were transferred to a nitrocellulose membrane and blotted with appropriate primary and secondary antibodies. Protein bands were detected by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). For quantiﬁcation of the amount of

e3 Cell Host & Microbe 25, 1–13.e1–e6, April 10, 2019 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

LCCL domain in the cochlea, we compared the LCCL domain from the PAO1-infected cochlea to the puriﬁed recombinant LCCL peptide (1 mg) by western blotting. Among 50 mg of lysate of the cochlea, LCCL domain comprised 1 mg, which represented 2% of the cochlea protein.

Immunostaining and Whole-mount ImmunoFluorescence Isolated cochleas were obtained by microdissection. Tissues were fixed by submersion in 4% formaldehyde at 4C overnight. After washing twice with PBS, the fixed temporal bones were decalcified for 24 h in 10% ethylenediaminetetraacetic acid (EDTA)/PBS. For paraffin sectioning and hematoxylin and eosin (H&E) staining, serial dehydration of the tissues was performed with ethanol and xylene. Then, the tissues were either embedded in paraffin for standard histological examination or dissected into smaller pieces and permeabilized with 0.1% Triton X-100 for whole-mount immunofluorescence. The paraffin blocks were sliced into 5 mm-thick sections in the mid-modiolar plane using a microtome (Leica Biosystems, Nussloch, Germany). Deparaffinization was performed on the sections as well as the whole-mounted tissues with a series of washes with xylene, ethanol, and PBS. After incubation in tris-sodium citrate at 95C for antigen-retrieval, the tissues were blocked with 10% donkey serum and incubated with target-specific primary and secondary antibodies at 4C overnight. The samples were then mounted with mounting solution (Sigma-Aldrich) and viewed under an LSM780 confocal microscope (Zeiss, Jena, Germany).

In situ Hybridization In situ hybridization was performed as described previously (Wilkinson et al., 1987). Briefly, the prehybridization treatment included steps to further fix the tissue, partially digest the sections with proteinase K to improve the probe’s access to the mRNA, and reduce non-specific probe binding by blocking the basic groups via acetylation. Tissue sections were incubated with the appropriate 3ss- labeled RNA probe (anti-Coch) in hybridization mixture at 55C overnight. The slides were washed under stringent conditions and treated with RNAase to remove unhybridized, non-specifically bound probe, followed by dehydration and drying. The slides were coated with Ilford K5 liquid emulsion for autoradiography and exposed for 3–4 weeks. After development, the sections were counter- stained with methyl green and mounted under a coverslip with DPX. The sections were examined by bright- and dark-field illumination.

Flow Cytometry Immune cells were isolated from the cochleas of Coch+/+ and Coch/ mice and sorted using a fluorescence-activated cell sorting (FACS) system as described previously (Ryu et al., 2017). In brief, dissected cochlea tissues were trypsinized at 37C for 11 min and mechanically dissociated in PBS with 2% FBS. Dissociated cells were then isolated with Percoll density gradient centrifugation. The isolated cells were rinsed in FACS buffer (1% fetal calf serum and 0.1% wt/vol sodium azide) on ice. Immune cells in the cochlea were counted by cell sorting using cluster of differentiation (CD)11b, CD11c, CD64, F4/80, and Ly6G antibodies and analyzed with a FACSverse BD flow cytometer (BD Biosciences, Sparks, MD, USA). For FACS analysis of cells from blood, blood sampling was performed via cardiac puncture. For bone marrow cell collection, bone marrow cells were isolated from mouse femur and tibia bone marrow. Red blood cells were lysed to remove background signals. The isolated cells were stained with antibodies against CD11b, Ly6G, and Ly6C to distinguish neutrophils and monocytes and analyzed with a FACS LSR II BD flow cytometer (BD Biosciences).

Enzyme-Linked Immunosorbent Assay To confirm that the multiplex enzyme-linked immunosorbent assay (ELISA) is as sensitive as traditional ELISA techniques, the tissue lysates were assayed with a DuoSet ELISA Development System kit from R&D Systems (Minneapolis, MN, USA) for single protein quantification. Expression of IL-1b, IL-6, monocyte chemoattractant protein (MCP)-1, KC (CXCL1), and macrophage inflammatory protein (MIP)-2 (CXCL2) was measured in the control and infected extracts. Target-specific antibodies were incubated overnight on medium binding Costar 96-well plates and rinsed three times in wash buffer. A total of 100 mL of mouse serum (and a 7-point dupli- cate standard curve) in reagent diluent was incubated for 2 h and washed three times with wash buffer. The wells were then incubated 2 h with biotinylated IL-1b, IL-6, MCP-1, or KC detection antibody, rinsed three times with wash buffer, reacted with streptavidin- horseradish peroxidase in reagent diluent for 20 min, washed three times with wash buffer, and incubated 20 min in a 1:1 substrate solution of hydrogen peroxide and tetramethylbenzidine (R&D Systems) to generate the blue colorimetric reaction. The plates were quenched and immediately read on a BioTek EL 800 plate reader at 450 and 540 nm.

Mouse Neutrophil Isolation

After euthanizing mice with CO2, the tibia and femur were separated and placed in RPMI 1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% FBS, 1% Penicillin-Streptomycin-Amphotericin (PSA) B Mixture (Lonza, Walkersville, MD, USA), and 2 mM EDTA at 4C. The bone marrow cells were obtained from tibia and femur and centrifuged at 1,500 rpm for 3 min at room temperature. Pellets were washed with PBS under the same conditions. Then erythrocytes were removed by incubation at room temperature for 5 min using ACK lysis buffer (Gibco). The erythrocyte-depleted bone marrow cells were suspended in PBS supplemented with 2% FBS, 1 mM EDTA. Neutrophils were puriﬁed by negative selection using the EasySep mouse neutrophil enrichment kit (Stemcell Technologies, Vancouver, BC, Canada) according to manufacturer’s instructions. After separation, neutrophils were re-suspended in the appropriate medium for the purpose.

Cell Host & Microbe 25, 1–13.e1–e6, April 10, 2019 e4 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

Scanning Electron Microscopy P. aeruginosa was cultured on coverslips with or without LCCL (1 mg/mL). Neutrophils (5 3 106 cells/mL) were re-suspended in RPMI 1640 medium supplemented with 10% FBS, 1% PSA and incubated in 24-well plates coated with 10 mg/mL of ﬁbronectin (Sigma) for 2 h at 37C with or without LCCL (1 mg per well). Then neutrophils were washed twice with pre-warmed PBS. The neutrophils and the bacteria were then immersed in 2% glutaraldehyde/paraformaldehyde in 0.1 M PBS, pH 7.4 for 6 h. After washing twice in 0.1 M PBS for 30 min each, the bacteria were post-ﬁxed with 1% OsO4 dissolved in 0.1 M PBS for 1.5 h and dehydrated in an ascending gradual series (50–100%) of ethanol. The bacteria were then treated with isoamyl acetate and subjected to critical point drying LEICA EM CPD300 (Vienna, Austria). Then, they were coated with Pt (5 nm) using an ion coater (LEICA EM ACE600) and examined and photo- graphed with a scanning electron microscope (FE-SEM; Merin, ZEISS).

Tissue Clearing and Immunostaining Anesthetized mice were perfused to remove whole blood from the entire body. The tissues were fixed by myocardial perfusion with 1X PBS and 4% formaldehyde, and the cochleae were removed by surgical exclusion. Tissues were further fixed by submersion in 4% formaldehyde at 4C overnight. After washing twice with PBS, the fixed temporal bones were decalcified for 24 h in 10% EDTA/ PBS (similar to the fixation steps listed in the Immunostaining and Whole-Mount Immunofluorescence section). The decalcified tissues were cleared using an Organ Clearing kit (Binaree, Seoul, Korea). Although the protocols were modified for small tissues (about 3mm3 3mm3 3 mm), each subsequent step followed that listed in the manufacturer’s protocol. To enhance tissue rigidity and preserve lipids and proteins, the samples were incubated at 4C while shaking for 12 h in fixing solution. Then, the cochlear lipids were removed and hydrated in detergent-based tissue clearing solution at 37C overnight while shaking. To remove the lipid/tissue clearing solutions, the cochleae were washed twice with washing solution at 4C for 6 h while shaking. Prior to immunostaining, the pre-cleared cochleae were blocked in 1X PBS containing 0.25% Triton X-100, 5% bovine serum albumin (BSA), and 0.01% sodium azide for 2 h at room temperature while shaking. To match the high refractive index (RI) of the tissue and render tissue transparency, the samples were incubated in mounting and storage solution at 37C overnight while shaking. Mounted samples were stored in the dark at room temperature until imaging.

Single Plane Illumination Microscopy Single plane illumination microscopy (SPIM) was performed with a Lightsheet Z.1 dual side illumination light sheet fluorescence microscope system (Zeiss). Laser lines (488, 561, and 640 nm) were used in single illumination mode. Cleared cochleae were immersed in mounting chamber with sample holder. Each tile was obtained in multitrack and z-stack mode using a Clr Plan-Neofluar 20X/1.0 objective lens, Corr nd=1.45. To generate separate czi and tiff files, tile stitching was performed using Arivis Vision 4D (Arivis AG, Munich, Germany). The Imaris v7.2.3 3D/4D image analysis software program (Bitplane, Oxford Instruments, Zurich, Switzerland) was used to process the 3D meta imaging data and analyze the cell counts and localization of the neutrophils or macrophages.

Auditory Function Test Auditory brainstem response (ABR) measurements were recorded as described previously (Jung et al., 2016). Brieﬂy, tests were conducted using an ABR workstation manufactured by Tucker Davis Technology (TDT) (Alachua, FL, USA). Properly anesthetized mice were placed on a heating pad (37C) in a soundproofed chamber. After electrode insertion, acoustic stimuli were applied through a receiver probe. The stimuli included 500-repeated click sounds or tone bursts with a 1 ms rise/fall time and a 5-ms plateau at fre- quencies of 4, 8, 16, and 32 kHz. The sound intensity started from a 90 dB SPL with 5 dB decreasing steps to the auditory threshold.

Biofilm Assay Biofilm formation assay was performed as previously described (O’Toole, 2011). Briefly, Pseudomonas aeruginosa was grown overnight in LB medium and diluted 1:100 into fresh medium, then 100 mL was added in a 96-well dish with four replicates. After incubation for 24 h at 37C, cells were poured out and the plate was washed twice with water. Then, 125 mL of a 0.4% solution of crystal violet in water was added, and after 15 min the plate was rinsed 3–4 times and dried overnight. For quantitative assays, 125 mL of 30% acetic acid was added to the plate to solubilize the crystal violet, transferred to a flat bottomed microtiter dish, and the absorbance at 550 nm was measured.

Transwell Assay LysM-GFP+/ mouse neutrophils were used for ﬂuorescent imaging and simple quantitation. Bone marrow cells were isolated from mouse femur and tibia bone marrow. Neutrophil isolation was performed as described above. Isolated neutrophils were added into 8.0 mm pore sized Matrigel- (5 mg/mL) (Corning, Armonk, NY, US) coated Transwell Inserts based on L-15 medium, and 1 mg of pu- riﬁed LCCL domain was dropped into the lower chamber. As a control, the lower chamber was also prepared in the absence of LCCL. After 2 h incubation, neutrophils were counted from the lower chamber to observe the effect of LCCL on neutrophil migration.

Liquid Chromatography-Tandem Mass Spectrometry Cleaved fragment (8–18 kDa) from heterologously expressed human cochlin in HEK293 cells was enriched by immunoprecipitation using an N-terminal cochlin antibody, purified, and loaded on a 1D gel. The band of cochlin was excised and digested in the 1D gel with sequencing grade, modified trypsin (Promega, Madison, WI, USA) as previously described (Bahk et al., 2004). Briefly, each e5 Cell Host & Microbe 25, 1–13.e1–e6, April 10, 2019 Please cite this article in press as: Jung et al., Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear, Cell Host & Microbe (2019), https://doi.org/10.1016/j.chom.2019.02.001

protein band was excised from the polyacrylamide electrophoresis gel, placed in a polypropylene tube (Eppendorf, Hamburg, Ger- many), and washed 4–5 times with 150 mL of 1:1 acetonitrile (CAN)/25 mM ammonium bicarbonate (ABC), pH 7.8. The gel slices were dehydrated by 100% CAN and then dried in a Speedvac concentrator (ThermoFischer, Waltham, USA). After reduction, alkylation, rehydration, trypsinization, tryptic peptides were dissolved in 8 mL of 5% (v/v) aqueous acetonitrile solution containing 0.1% (v/v) formic acid for mass spectrometric (MS) analysis. The resulting tryptic peptides were separated and analyzed using reversed phase capillary high performance liquid chromatography directly coupled to a Finnigan LCQ ion-trap mass spectrometer (LC-MS/MS) (Zuo et al., 2001). Both the 0.1 3 20-mm trapping and the 0.075 3 130-mm resolving columns were packed with Vydac 218MS low trifluoroacetic acid C18 beads (5 mm diameter, 300 A˚ pore size) and placed in-line. The peptides were bound to the trapping col- umn for 10 min in 5% (v/v) aqueous ACN containing 0.1% (v/v) formic acid, and the bound peptides were eluted with a 50-min gradient of 5%–80% (v/v) ACN containing 0.1% (v/v) formic acid at a flow rate of 0.2 mL min-1. For tandem mass spectrometry, the full mass scan range mode was an m/z of 450–2000 Da. After determination of the charge states of an ion using zoom scans, product ion spectra were acquired in MS/MS mode with a relative collision energy of 55%. The individual MS/MS spectra were pro- cessed using the TurboSEQUEST software (Thermo Quest, San Jose, CA, USA). The generated peak list files were used to query either the MSDB or NCBI database using the MASCOT program (http://www.matrixscience.com). Modifications of methionine and cysteine (carbamidomethyl [C], deamidated [NQ], oxidation [M]) with a peptide mass tolerance of 2 Da, MS/MS ion mass tolerance of 1 Da, allowance of missed cleavage of 1, and charge states of +1, +2, and +3, were all taken into account. Only significant hits as defined by MASCOT probability analysis were considered initially.

QUANTIFICATION AND STATISTICAL ANALYSIS

All statistical analyses were performed with SPSS software for PC, version 21 (SPSS, Chicago, IL, USA). The results of multiple experiments are presented in figures as the means ± standard error of the mean (SEM). For each experiment, the number of replicates or other relevant informations for assessing the precision and accuracy of the quantified measurements are included in the corresponding figure legend. Statistical comparisons were performed using a Student’s t-test, paired t-test, or an analysis of variance (ANOVA) followed by the Bonferroni multiple comparison test, where appropriate. A p < 0.05 was considered statistically significant.

DATA AND SOFTWARE AVAILABILITY

The data and the codes of this study are available from the corresponding author upon reasonable request.

Cell Host & Microbe 25, 1–13.e1–e6, April 10, 2019 e6