ADAR1 prevents autoinflammation by suppressing spontaneous ZBP1 activation.

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)1. In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutières syndrome (AGS)2. In mice, complete loss of ADAR1 activity is embryonically lethal3-6, and mutations similar to those found in patients with AGS cause autoinflammation7-12. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR3,7-10,13,14. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function.

MLKL Reveals Its Friendly Face: A Role in Nerve Regeneration.

In this issue of Molecular Cell, Ying et al. (2018) report on a novel function of MLKL in sciatic nerve regeneration after injury through myelin sheath destabilization. This function of MLKL is independent of necroptosis execution and requires phosphorylation at MLKL-S441.

RIP kinases at the crossroads of cell death and survival.

Protein kinases of the receptor interacting protein (RIP) family collaborate with death receptor proteins to regulate cell death. Recent studies (Cho et al., 2009; He et al., 2009; Zhang et al., 2009) reveal that the RIP3 kinase functions with RIP1 at the crossroads of apoptosis, necroptosis, and cell survival.

Functions of the RIP kinase family members in the skin.

The receptor interacting protein kinases (RIPK) are a family of serine/threonine kinases that are involved in the integration of various stress signals. In response to several extracellular and/or intracellular stimuli, RIP kinases engage signaling cascades leading to the activation of NF-κB and mitogen-activated protein kinases, cell death, inflammation, differentiation and Wnt signaling and can have kinase-dependent and kinase-independent functions. Although it was previously suggested that seven RIPKs are part of the RIPK family, phylogenetic analysis indicates that there are only five genuine RIPKs. RIPK1 and RIPK3 are mainly involved in controlling and executing necroptosis in keratinocytes, while RIPK4 controls proliferation and differentiation of keratinocytes and thereby can act as a tumor suppressor in skin. Therefore, in this review we summarize and discuss the functions of RIPKs in skin homeostasis as well as the signaling pathways involved.

Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding.

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and Kd values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.

Immunodominant AH1 Antigen-Deficient Necroptotic, but Not Apoptotic, Murine Cancer Cells Induce Antitumor Protection.

Immunogenic cell death (ICD) occurs when a dying cell releases cytokines and damage-associated molecular patterns, acting as adjuvants, and expresses Ags that induce a specific antitumor immune response. ICD is studied mainly in the context of regulated cell death pathways, especially caspase-mediated apoptosis marked by endoplasmic reticulum stress and calreticulin exposure and, more recently, also in relation to receptor-interacting protein kinase-driven necroptosis, whereas unregulated cell death like accidental necrosis is nonimmunogenic. Importantly, the murine cancer cell lines used in ICD studies often express virally derived peptides that are recognized by the immune system as tumor-associated Ags. However, it is unknown how different cell death pathways may affect neoepitope cross-presentation and Ag recognition of cancer cells. We used a prophylactic tumor vaccination model and observed that both apoptotic and necroptotic colon carcinoma CT26 cells efficiently immunized mice against challenge with a breast cancer cell line that expresses the same immunodominant tumor Ag, AH1, but only necroptotic CT26 cells would mount an immune response against CT26-specific neoepitopes. By CRISPR/Cas9 genome editing, we knocked out AH1 and saw that only necroptotic CT26 cells were still able to protect mice against tumor challenge. Hence, in this study, we show that endogenous AH1 tumor Ag expression can mask the strength of immunogenicity induced by different cell death pathways and that upon knockout of AH1, necroptosis was more immunogenic than apoptosis in a prophylactic tumor vaccination model. This work highlights necroptosis as a possible preferred ICD form over apoptosis in the treatment of cancer.

Plasma membrane perforation by GSDME during apoptosis-driven secondary necrosis.

Secondary necrosis has long been perceived as an uncontrolled process resulting in total lysis of the apoptotic cell. Recently, it was shown that progression of apoptosis to secondary necrosis is regulated by Gasdermin E (GSDME), which requires activation by caspase-3. Although the contribution of GSDME in this context has been attributed to its pore-forming capacity, little is known about the kinetics and size characteristics of this. Here we report on the membrane permeabilizing features of GSDME by monitoring the influx and efflux of dextrans of different sizes into/from anti-Fas-treated L929sAhFas cells undergoing apoptosis-driven secondary necrosis. We found that GSDME accelerates cell lysis measured by SYTOX Blue staining but does not affect the exposure of phosphatidylserine on the plasma membrane. Furthermore, loss of GSDME expression clearly hampered the influx of fluorescently labeled dextrans while the efflux happened independently of the presence or absence of GSDME expression. Importantly, both in- and efflux of dextrans were dependent on their molecular weight. Altogether, our results demonstrate that GSDME regulates the passage of compounds together with other plasma membrane destabilizing subroutines.

Dual face apoptotic machinery: from initiator of apoptosis to guardian of necroptosis.

In this issue of Immunity, Bonnet et al. (2011) show that skin-specific ablation of the adaptor protein FADD sensitizes keratinocytes to RIPK3-dependent necrotic cell death, which leads to severe skin inflammation.

RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome.

Engagement of tumor necrosis factor receptor 1 signals two diametrically opposed pathways: survival-inflammation and cell death. An additional switch decides, depending on the cellular context, between caspase-dependent apoptosis and RIP kinase (RIPK)-mediated necrosis, also termed necroptosis. We explored the contribution of both cell death pathways in TNF-induced systemic inflammatory response syndrome (SIRS). Deletion of apoptotic executioner caspases (caspase-3 or -7) or inflammatory caspase-1 had no impact on lethal SIRS. However, deletion of RIPK3 conferred complete protection against lethal SIRS and reduced the amounts of circulating damage-associated molecular patterns. Pretreatment with the RIPK1 kinase inhibitor, necrostatin-1, provided a similar effect. These results suggest that RIPK1-RIPK3-mediated cellular damage by necrosis drives mortality during TNF-induced SIRS. RIPK3 deficiency also protected against cecal ligation and puncture, underscoring the clinical relevance of RIPK kinase inhibition in sepsis and identifying components of the necroptotic pathway that are potential therapeutic targets for treatment of SIRS and sepsis.

RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis.

Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.

Synthesis and evaluation of novel benzotropolones as Atg4B inhibiting autophagy blockers.

Autophagy is an intracellular degradation/recycling pathway that provides nutrients and building blocks to cellular metabolism and keeps the cytoplasm clear of obsolete proteins and organelles. During recent years, dysregulated autophagy activity has been reported to be a characteristic of many different disease types, including cancer and neurodegenerative disorders. This has created a strong case for development of autophagy modulating compounds as potential treatments for these diseases. Inhibitors of autophagy have been proposed as a therapeutic intervention in, e.g., advanced cancer, and inhibiting the cysteine protease Atg4B has been put forward as a main strategy to block autophagy. We recently identified and demonstrated -both in vitro and in vivo - that compounds with a benzotropolone basic structure targeting Atg4B, can significantly slow down tumor growth and potentiate the effect of classical chemotherapy. In this study we report the synthesis and inhibition profile of new benzotropolone derivatives with additional structural modifications at 6 different positions. To obtain a solid inhibition profile, all compounds were evaluated on three levels, including two cell-based assays to confirm autophagy and intracellular Atg4B inhibition and an SDS-PAGE-based experiment to assess in vitro Atg4B affinity. Several molecules with a promising profile were identified.

RIPK4 activity in keratinocytes is controlled by the SCFß-TrCP ubiquitin ligase to maintain cortical actin organization.

RIPK4 is a key player in epidermal differentiation and barrier formation. RIPK4 signaling pathways controlling keratinocyte proliferation and differentiation depend on its kinase activity leading to Dvl2, Pkp1 and IRF6 phosphorylation and NF-κB activation. However, the mechanism regulating RIPK4 activity levels remains elusive. We show that cultured keratinocytes display constitutive active phosphorylated RIPK4 while PKC signaling can trigger RIPK4 activation in various non-keratinocyte cell lines, in which RIPK4 is present in a non-phosphorylated state. Interestingly, we identified the SCFß-TrCP ubiquitin E3 ligase complex responsible for regulating the active RIPK4 protein level. The SCFß-TrCP complex binds to a conserved phosphodegron motif in the intermediate domain of RIPK4, subsequently leading to K48-linked ubiquitinylation and degradation. The recruitment of ß-TrCP is dependent on RIPK4 activation and trans-autophosphorylation. ß-TrCP knock-down resulted in RIPK4-dependent formation of actin stress fibers, cell scattering and increased cell motility, suggesting that tight control of RIPK4 activity levels is crucial to maintain cell shape and behavior in keratinocytes.

The IL-33/ST2 axis is crucial in type 2 airway responses induced by Staphylococcus aureus-derived serine protease-like protein D.

BACKGROUND: Chronic airway inflammatory diseases, such as chronic rhinosinusitis with nasal polyps and asthma, show increased nasal Staphylococcus aureus colonization. Staphylococcus aureus-derived serine protease-like protein (Spl) D and other closely related proteases secreted by S aureus have recently been identified as inducers of allergic asthma in human subjects and mice, but their mechanism of action is largely unknown. OBJECTIVE: We investigated the role of recombinant SplD in driving TH2-biased responses and IgE formation in a murine model of allergic asthma. METHODS: Allergic asthma was induced in C57BL/6 J wild-type mice, Toll-like receptor (TLR) 4 knockout (Tlr4-/-) mice, and recombination-activating gene (Rag2) knockout (Rag2-/-) mice by means of repeated intratracheal applications of SplD. Inflammatory parameters in the airways were assessed by means of flow cytometry, ELISA, Luminex, and immunohistochemistry. Serum SplD-specific IgE levels were analyzed by using ELISA. RESULTS: We observed that repeated intratracheal exposure to SplD led to IL-33 and eotaxin production, eosinophilia, bronchial hyperreactivity, and goblet cell hyperplasia in the airways. Blocking IL-33 activity with a soluble ST2 receptor significantly decreased the numbers of eosinophils, IL-13+ type 2 innate lymphoid cells and IL-13+CD4+ T cells and IL-5 and IL-13 production by lymph node cells but had no effect on IgE production. SplD-induced airway inflammation and IgE production were largely dependent on the presence of the functional adaptive immune system and independent of TLR4 signaling. CONCLUSION: The S aureus-derived protein SplD is a potent allergen of S aureus and induces a TH2-biased inflammatory response in the airways in an IL-33-dependent but TRL4-independent manner. The soluble ST2 receptor could be an efficient strategy to interfere with SplD-induced TH2 inflammation but does not prevent the allergic sensitization.

Delivery of Mixed-Lineage Kinase Domain-Like Protein by Vapor Nanobubble Photoporation Induces Necroptotic-Like Cell Death in Tumor Cells.

Modern molecular medicine demands techniques to efficiently deliver molecules directly into mammalian cells. As proteins are the final mediators of most cellular pathways, efficient intracellular protein delivery techniques are highly desired. In this respect, photoporation is a promising recent technique for the delivery of proteins directly into living cells. Here, we show the possibility to deliver a model saccharide (FD70) and a model protein (FITC-BSA) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively. Next, we delivered the mixed-lineage kinase domain-like (MLKL) protein, the most terminal mediator of necroptosis currently known, and caspase-8 and -3 protein, which are important proteins in the initiation and execution of apoptosis. A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively, was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery. Transduction of purified recombinant MLKL by photoporation resulted in rapid cell death characterized by cell swelling and cell membrane rupture, both hallmarks of necroptosis. As necroptosis has been identified as a type of cell death with immunogenic properties, this is of interest to anti-cancer immunotherapy. On the other hand, transduction of purified recombinant active caspase-3 or -8 into the tumor cells resulted in rapid cell death preceded by membrane blebbing, which is typical for apoptosis. Our results suggest that the type of cell death of tumor cells can be controlled by direct transduction of effector proteins that are involved in the executioner phase of apoptosis or necroptosis.

Feasibility study for clinical application of caspase-3 inhibitors in Pemphigus vulgaris.

The potentially severe side effects of systemic corticosteroids and immunosuppressants used in Pemphigus vulgaris (PV) call for novel therapeutic approaches. In this context, pharmacological inhibition of major pathogenic signalling effectors represents a promising alternative. However, we have also shown that overinhibition of effectors required for epidermal homeostasis can exacerbate PV pathophysiology implicating transepidermal keratinocyte fragility. A feedforward target validation therefore preferentially includes studies on knockout mouse models. We previously reported on successful amelioration of PV blisters following inhibition of non-apoptotic, low-level caspase-3. Here, we use conditional, keratinocyte-specific caspase-3-deficient mice (casp3EKO ) to demonstrate (i) absence of keratinocyte fragility upon injection of the potent Dsg3-specific antibody AK23 and (ii) amelioration of blistering on the background of known signalling effectors. Our results provide the experimental proof of concept justifying translation of the caspase-3 inhibitor approach into PV clinical trials.

CDK4 T172 phosphorylation is central in a CDK7-dependent bidirectional CDK4/CDK2 interplay mediated by p21 phosphorylation at the restriction point.

Cell cycle progression, including genome duplication, is orchestrated by cyclin-dependent kinases (CDKs). CDK activation depends on phosphorylation of their T-loop by a CDK-activating kinase (CAK). In animals, the only known CAK for CDK2 and CDK1 is cyclin H-CDK7, which is constitutively active. Therefore, the critical activation step is dephosphorylation of inhibitory sites by Cdc25 phosphatases rather than unrestricted T-loop phosphorylation. Homologous CDK4 and CDK6 bound to cyclins D are master integrators of mitogenic/oncogenic signaling cascades by initiating the inactivation of the central oncosuppressor pRb and cell cycle commitment at the restriction point. Unlike the situation in CDK1 and CDK2 cyclin complexes, and in contrast to the weak but constitutive T177 phosphorylation of CDK6, we have identified the T-loop phosphorylation at T172 as the highly regulated step determining CDK4 activity. Whether both CDK4 and CDK6 phosphorylations are catalyzed by CDK7 remains unclear. To answer this question, we took a chemical-genetics approach by using analogue-sensitive CDK7(as/as) mutant HCT116 cells, in which CDK7 can be specifically inhibited by bulky adenine analogs. Intriguingly, CDK7 inhibition prevented activating phosphorylations of CDK4/6, but for CDK4 this was at least partly dependent on its binding to p21 (cip1) . In response to CDK7 inhibition, p21-binding to CDK4 increased concomitantly with disappearance of the most abundant phosphorylation of p21, which we localized at S130 and found to be catalyzed by both CDK4 and CDK2. The S130A mutation of p21 prevented the activating CDK4 phosphorylation, and inhibition of CDK4/6 and CDK2 impaired phosphorylations of both p21 and p21-bound CDK4. Therefore, specific CDK7 inhibition revealed the following: a crucial but partly indirect CDK7 involvement in phosphorylation/activation of CDK4 and CDK6; existence of CDK4-activating kinase(s) other than CDK7; and novel CDK7-dependent positive feedbacks mediated by p21 phosphorylation by CDK4 and CDK2 to sustain CDK4 activation, pRb inactivation, and restriction point passage.

Interaction of Mycobacterium leprae with the HaCaT human keratinocyte cell line: new frontiers in the cellular immunology of leprosy.

Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae affecting the skin and peripheral nerves. Despite M. leprae invasion of the skin and keratinocytes importance in innate immunity, the interaction of these cells in vitro during M. leprae infection is poorly understood. Conventional and fluorescence optical microscopy, transmission electronic microscopy, flow cytometry and ELISA were used to study the in vitro interaction of M. leprae with the HaCaT human keratinocyte cell line. Keratinocytes uptake of M. leprae is described, and modulation of the surface expression of CD80 and CD209, cathelicidin expression and TNF-α and IL-1ß production of human keratinocytes are compared with dendritic cells and macrophages during M. leprae interaction. This study demonstrated that M. leprae interaction with human keratinocytes enhanced expression of cathelicidin and greatly increased TNF-α production. The highest spontaneous expression of cathelicidin was by dendritic cells which are less susceptible to M. leprae infection. In contrast, keratinocytes displayed low spontaneous cathelicidin expression and were more susceptible to M. leprae infection than dendritic cells. The results show, for the first time, an active role for keratinocytes during infection by irradiated whole cells of M. leprae and the effect of vitamin D on this process. They also suggest that therapies which target cathelicidin modulation may provide novel approaches for treatment of leprosy.

Simultaneous targeting of IL-1 and IL-18 is required for protection against inflammatory and septic shock.

RATIONALE: Sepsis is one of the leading causes of death around the world. The failure of clinical trials to treat sepsis demonstrates that the molecular mechanisms are multiple and are still insufficiently understood. OBJECTIVES: To clarify the long disputed hierarchical contribution of several central inflammatory mediators (IL-1ß, IL-18, caspase [CASP] 7, CASP1, and CASP11) in septic shock and to explore their therapeutic potential. METHODS: LPS- and tumor necrosis factor (TNF)-induced lethal shock, and cecal ligation and puncture (CLP) were performed in genetically or pharmacologically targeted mice. Body temperature and survival were monitored closely, and plasma was analyzed for several markers of cellular disintegration and inflammation. MEASUREMENTS AND MAIN RESULTS: Interestingly, deficiency of both IL-1ß and IL-18 additively prevented LPS-induced mortality. The detrimental role of IL-1ß and IL-18 was confirmed in mice subjected to a lethal dose of TNF, or to a lethal CLP procedure. Although their upstream activator, CASP1, and its amplifier, CASP11, are considered potential therapeutic targets because of their crucial involvement in endotoxin-induced toxicity, CASP11- or CASP1/11-deficient mice were not, or hardly, protected against a lethal TNF or CLP challenge. In line with our results obtained in genetically deficient mice, only the combined neutralization of IL-1 and IL-18, using the IL-1 receptor antagonist anakinra and anti-IL-18 antibodies, conferred complete protection against endotoxin-induced lethality. CONCLUSIONS: Our data point toward the therapeutic potential of neutralizing IL-1 and IL-18 simultaneously in sepsis, rather than inhibiting the upstream inflammatory caspases.

Cell death by cornification.

Epidermal keratinocytes undergo a unique form of terminal differentiation and programmed cell death known as cornification. Cornification leads to the formation of the outermost skin barrier, i.e. the cornified layer, as well as to the formation of hair and nails. Different genes are expressed in coordinated waves to provide the structural and regulatory components of cornification. Differentiation-associated keratin intermediate filaments form a complex scaffold accumulating in the cytoplasm and, upon removal of cell organelles, fill the entire cell interior mainly to provide mechanical strength. In addition, a defined set of proteins is cross-linked by transglutamination in the cell periphery to form the so-called cornified envelope. Extracellular modifications include degradation of the tight linkages between corneocytes by excreted proteases, which allows corneocyte shedding by desquamation, and stacking and modification of the excreted lipids that fill the intercellular spaces between corneocytes to provide a water-repellant barrier. In hard skin appendages such as hair and nails these tight intercorneocyte connections remain permanent. Various lines of evidence exist for a role of organelle disintegration, proteases, nucleases, and transglutaminases contributing to the actual cell death event. However, many mechanistic aspects of kearatinocyte death during cornification remain elusive. Importantly, it has recently become clear that keratinocytes activate anti-apoptotic and anti-necroptotic pathways to prevent premature cell death during terminal differentiation. This review gives an overview of the current concept of cornification as a mode of programmed cell death and the anti-cell death mechanisms in the epidermis that secure epidermal homeostasis. This article is part of a Special Section entitled: Cell Death Pathways.

The skin microbiome of caspase-14-deficient mice shows mild dysbiosis.

Caspase-14, an important proteinase involved in filaggrin catabolism, is mainly active in terminally differentiating keratinocytes, where it is required for the generation of skin natural moisturizing factors (NMFs). Consequently, caspase-14 deficient epidermis is characterized by reduced levels of NMFs such as urocanic acid and 2-pyrrolidone-5-carboxylic acid. Patients suffering from filaggrin deficiency are prone to develop atopic dermatitis, which is accompanied with increased microbial burden. Among several reasons, this effect could be due to a decrease in filaggrin breakdown products. In this study, we found that caspase-14(-/-) mice show enhanced antibacterial response compared to wild-type mice when challenged with bacteria. Therefore, we compared the microbial communities between wild-type and caspase-14(-/-) mice by sequencing of bacterial 16S ribosomal RNA genes. We observed that caspase-14 ablation leads to an increase in bacterial richness and diversity during steady-state conditions. Although both wild-type and caspase-14(-/-) skin were dominated by the Firmicutes phylum, the Staphylococcaceae family was reduced in caspase-14(-/-) mice. Altogether, our data demonstrated that caspase-14 deficiency causes the imbalance of the skin-resident bacterial communities.