Graphical abstract

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Highlights

     * •
       A small molecule IAAP ameliorates LPS-induced inflammatory
       responses and ALI/ARDS
     * •
       IAAP interacts with and suppresses the bioactivity of CTSL
     * •
       CTSL is upregulated in BALF of patients with ARDS and the murine
       model of ALI
     * •
       CTSL degrades the deubiquitinase A20 and promotes the activation of
       NF-κB signaling
     __________________________________________________________________

   Biological sciences; Molecular biology; Molecular interaction

Introduction

   Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a
   fatal and undertreated condition characterized by increased pulmonary
   vascular permeability, massive inflammation, pulmonary edema, and
   refractory hypoxia. Clinically, it manifests as hypoxemia with
   bilateral opacities on chest radiography, in addition to reduced lung
   compliance and enhanced physiological dead space.[70]^1^,[71]^2 Studies
   have shown that the incidence of ALI/ARDS is 70/100 000 and 59/100 000
   annually, respectively, with a case fatality rate of approximately
   40%.[72]^3 Furthermore, the coronavirus of 2019 (COVID-19) pandemic has
   resulted in 6 million deaths worldwide, primarily due to complications
   from severe acute respiratory syndrome coronavirus 2
   (SARS-CoV-2)-associated ARDS, supporting the urgent need to better
   understand its molecular pathogenesis.[73]^4 To date, there are
   relatively few treatments available for ALI/ARDS, and the cornerstone
   of management is mechanical ventilation; drug therapy may differ
   depending on the individual patient and the inciting
   cause.[74]^5^,[75]^6 The proposed therapeutic drugs include common
   chemical drugs, antibiotics, and biomacromolecular drugs composed of
   proteins, polypeptides, and genetic materials (such as
   RNA).[76]^7^,[77]^8^,[78]^9^,[79]^10 However, no drug has been shown to
   significantly reduce the mortality rate in ALI/ARDS in clinical
   trials.[80]^5^,[81]^11^,[82]^12 Therefore, it is of great significance
   to identify new intervention targets for the clinical prevention and
   treatment of ALI/ARDS.

   ALI/ARDS can be caused by various pulmonary insults (such as pneumonia
   and aspiration) or non-pulmonary causes (such as sepsis, pancreatitis,
   and trauma).[83]^13 Lipopolysaccharide (LPS) is the primary component
   of the outer membrane of Gram-negative bacteria and is capable of
   inducing a wide range of infections, such as severe pneumonia and
   septicemia.[84]^14 Signaling also activates the downstream nuclear
   factor kappa-B (NF-κB) and augments inflammatory mediators.[85]^15
   Hence, LPS has emerged as a clinically relevant model for
   ALI/ARDS.[86]^16 The NF-κB family is a key regulator of innate and
   adaptive immune responses.[87]^17 In ALI/ARDS, LPS activates canonical
   NF-κB activation, which typically involves K63-polyubiquitinated NF-κB
   essential modulator (NEMO) and subsequent phosphorylation of
   IκBα.[88]^18^,[89]^19

   The lysosome is an active metabolic site in cells and the primary
   organelle for the decomposition and recycling of various biological
   macromolecules. Lysosomes are involved in a variety of life processes,
   including gene regulation, signal transduction, energy metabolism, and
   immunity, and are at the center of complex networks that regulate the
   stability of the internal environment of cells and
   organisms.[90]^20^,[91]^21^,[92]^22^,[93]^23 Cathepsins are a family of
   multifunctional proteases that are synthesized and transported as
   zymogens into lysosomes and are activated by cleavage in the acidic
   environment of the lysosome.[94]^22^,[95]^24^,[96]^25 Studies have
   shown that cathepsins are crucial for antigen presentation,
   inflammasome signaling, vascular remodeling, and protein cleavage
   processing.[97]^26^,[98]^27^,[99]^28 Cathepsin L (CTSL) plays an
   important role in regulating antigen proteolysis and processing the
   MHC-bound invariant chain (li).[100]^29^,[101]^30 The p41 splice
   variant of MHC class II-associated Ii binds noncovalently to the active
   site of CTSL and protects mature-CTSL from
   degradation.[102]^31^,[103]^32 Recent studies have shown that CTSL is
   highly expressed in the respiratory system, and CTSL inhibitors, such
   as SID 26681509 and E64d, reduce live viral infection of ex vivo lung
   tissues of both human donors and human ACE2-transgenic
   mice.[104]^33^,[105]^34 Eosinophil-derived cathepsin L promotes
   pulmonary matrix destruction and emphysema by degrading the
   extracellular matrix.[106]^35 However, whether lysosomal CTSL is
   involved in the development of ALI/ARDS remains unclear.

   In the present study, we explored the effects and mechanisms of IAAP on
   LPS-induced responses. Our findings revealed that LPS facilitated
   CTSL-mediated degradation of A20, thereby promoting the activation of
   NF-κB signaling, whereas IAAP suppresses LPS-induced inflammatory
   responses by targeting and inhibiting lysosomal CTSL.

Results

IAAP ameliorates lipopolysaccharide-induced inflammatory response in vitro
and acute lung injury in vivo

   In previous studies, heterocyclic compounds containing indoles or
   pyrimidines were shown to have significant anti-inflammatory
   effects.[107]^36^,[108]^37 We designed and synthesized IAAP using
   4-indole-2-arylaminopyrimidine as the core skeleton.[109]^38 Based on
   classical drug design principles, minimum change principle, and
   bioisosterism, we improved the hydrophilicity and enhanced the
   biological activity of IAAP by replacing the fluorine atoms with
   various amino substituents at position four of the phenyl ring. The
   molecular structure of IAAP is shown in [110]Figure 1A. We first
   examined the cell cytotoxicity of IAAP in BMDM, HBE, and THP-1 cells
   using MTT assays ([111]Figure S1A), and IAAP showed no obvious
   cytotoxicity (cell survival rates ≥70%). To determine the optimal
   concentration of IAAP in vitro and in vivo, the protein levels of IL-6
   induced by LPS were measured after treatment with different
   concentrations of IAAP in HBE, THP-1, and BMDM ([112]Figures S1B–S1D),
   and the anti-inflammatory effects of different concentrations of IAAP
   in LPS-induced ALI in mice were evaluated ([113]Figures S1E–S1G). Then
   the protein levels of IL-6, IL-8, CXCL1, and CXCL2 induced by LPS were
   significantly downregulated after treatment with 5 μM IAAP in BMDM,
   HBE, and THP-1 cells, respectively ([114]Figures 1B–1D). In a murine
   model of intratracheal LPS-induced inflammation and ALI/ARDS, IAAP
   (20 mg/kg) led to a significant reduction in total cell counts,
   neutrophil numbers, and protein concentrations in the bronchoalveolar
   lavage fluid (BALF) compared to LPS-treated mice ([115]Figures 1E and
   1F). This protective effect was also confirmed by the reduced levels of
   chemokines and the pro-inflammatory cytokines CXCL1, CXCL2, and IL6 in
   the BALF ([116]Figure 1G). Histopathological examination revealed that
   IAAP significantly reduced the severity of lung inflammation and damage
   ([117]Figure 1H). As expected, the mice treated with IAAP exhibited
   significantly higher survival rates than the control group after LPS
   administration ([118]Figure 1I). These results indicate that IAAP
   ameliorated the LPS-induced inflammatory response in vitro and ALI
   in vivo.

Figure 1.

   [119]Figure 1
   [120]Open in a new tab

   IAAP ameliorates LPS-induced inflammatory response in vitro and ALI
   in vivo

   (A) The chemical structure of IAAP.

   (B) BMDM were stimulated with LPS (100 ng/mL) or treated with IAAP
   (5 μM) for 6 h. Cells were then harvested to analyze secreted IL-6,
   CXCL1, and CXCL2 proteins in cell culture supernatants using ELISA, n =
   3 each group.

   (C) HBE were stimulated with LPS (100 μg/mL), or treated with IAAP
   (5 μM) for 24 h. Cells were then harvested to analyze IL-6 and IL-8
   proteins in cell culture supernatants using ELISA, n = 3 each group.

   (D) THP-1 were stimulated with LPS (100 ng/mL), or treated with IAAP
   (5 μM) for 6 h. Cells were then harvested for analyzing secreted IL-6
   and IL-8 proteins in cell culture supernatants using ELISA, n = 3 each
   group.

   (E) Mice were treated by intraperitoneal injection with IAAP (20 mg/kg)
   1h before pentobarbital injection and then with LPS (in 50 μL saline)
   at a dose of 5 mg/kg or NS though intratracheal administration. Mice
   were sacrificed 24 h after LPS administration for analysis. Total and
   differential cell counts in the BALF from mice: n (NS + DMSO) = 3, n
   (LPS + DMSO) = 5, n (IAAP) = 3, n (IAAP + LPS) = 5.

   (F) Total protein concentration in the BALF.

   (G) Expression of IL6, CXCL1, and CXCL2 in the BALF.

   (H) Representative images of lung tissue with HE staining 24 h post-LPS
   challenge and histological inflammatory scores. Scale bar: 200 μm.

   (I) For the survival assay, mice were intratracheally treated with LPS
   (20 mg/kg) on day 0. Mice received an intraperitoneal injection of IAAP
   (20 mg/kg) or PBS 1h before intratracheal instillation on day 0 and
   were injected with drugs twice on day 3. Survival rates of mice after
   the intratracheal administration of LPS with or without IAAP
   intraperitoneal injection for 24 h. n (DMSO + LPS) = 17, n (IAAP +
   LPS) = 18. DMSO was used as the vehicle control. All the quantitative
   data are presented as mean ± SEM and differences were identified using
   one-way ANOVA. The log rank (Mantel-Cox) test was used to analyze the
   survival rates. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

IAAP impairs lysosomal degradation activity leading to the excessive
accumulation of autolysosomes

   To investigate the underlying mechanisms of IAAP in LPS-induced
   inflammation, a biotin-labeled IAAP was designed and synthesized. We
   performed co-IP experiments with IAAP-biotin and used
   immunoprecipitation and liquid chromatography-tandem mass spectrometry
   (LC-MS/MS) to qualitatively analyze the protein molecules that interact
   with IAAP-biotin. LC-MS/MS revealed that IAAP-biotin could bind to 1913
   proteins. After excluding 1744 proteins that bound non-specifically to
   biotin or Streptomycin C1, we screened 169 proteins that
   were specifically bound to IAAP. The full LC-MS/MS results are
   presented in [121]Data S2. The identified proteins mapped to 169 genes.
   Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
   analysis of these genes revealed that IAAP primarily acts on steps in
   the autophagy-lysosome process ([122]Figure 2A).

Figure 2.

   [123]Figure 2
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   IAAP impairs lysosomal degradation activity leading to the excessive
   accumulation of autolysosomes

   (A) List of IAAP-related pathways. HBE cells were treated with biotin
   (10 μM), IAAP (10 μM), or IAAP-biotin (10 μM) for 24 h and then
   harvested for Co-IP. The resulting Co-IP protein solutions were used
   for protein identification by LC-MS/MS. LC-MS/MS results were used for
   KEGG pathway enrichment analysis.

   (B) HBE, BMDM, and THP-1 were treated with IAAP (5 μM) for 6 h, the
   levels of LC3B and SQSTM1 were examined using Western blotting.

   (C) HBE were treated with IAAP (5 μM) or Baf A1 (5 nM) for 6 h.
   Cellular LC3B and SQSTM1 levels were analyzed using Western blotting.

   (D) HBE expressing GFP-RFP-LC3B were treated with IAAP (5 μM) or Baf A1
   (5 nM) for 6 h and imaged using confocal microscopy. Scale bar: 10 μm.

   (E) HBE was treated with IAAP (5 μM) or Baf A1 (5 nM) for 6 h, then the
   cells were imaged using electron microscopy. AV, autophagic
   vacuole/autophagosome; AL, autolysosome; Lyso, lysosome. Scale bar:
   2 μm.

   (F) HBE were treated with IAAP (5 μM) for 6 h, and the levels of EEA1,
   LAMP1, Mature-CTSD, and V-ATPase D were examined using Western
   blotting.

   (G) Western blot analysis of EGFR at the indicated time points after
   EGF incubation in cells treated with IAAP (5 μM) or Baf A1 (5 nM) for 6
   h. DMSO served as the vehicle control.

   To determine whether IAAP regulates LPS-induced airway inflammation
   through autophagy-lysosome signaling, we evaluated the levels of
   autophagic proteins in different cell lines. When autophagy is
   activated, LC3B accumulates as an autophagy marker for autophagosome
   formation and the autophagy substrate SQSTM1 is preferentially
   degraded. The results revealed that LC3B expression was significantly
   increased in IAAP-treated cells ([125]Figures 2B and [126]S2A). In
   contrast, IAAP increased instead of decreased SQSTM1 protein levels in
   a dose-dependent manner, suggesting that autophagic degradation was
   inhibited by IAAP treatment ([127]Figure 2B). Upon Bafilomycin A1
   (Baf A1)-treatment, autophagic proteins reached levels similar to those
   of IAAP treatment alone, and there were few synergistic or additive
   effects between Baf A1 and IAAP ([128]Figure 2C), suggesting that the
   accumulation of LC3B and SQSTM1 induced by IAAP resulted from decreased
   degradation of the autophagosome. Next, we monitored autophagy flux
   using the GFP-RFP-LC3B plasmid. Both red and green fluorescence was
   observed in Baf A1-treated cells, indicating autophagosome
   accumulation. Interestingly, IAAP treatment resulted in
   the accumulation of red dots with few punctate green fluorescence
   ([129]Figure 2D). Transmission electron microscopy (TEM) revealed that
   IAAP triggered a significant accumulation of autolysosomes, displaying
   relatively large-sized single-membrane vesicles with visible
   cytoplasmic contents, whereas Baf A1 induced accumulation of
   autophagosomes ([130]Figure 2E).

   We assessed lysosomal biosynthesis and maturation. Lysosomes are
   progressively acidified by the action of vacuolar-type H + -ATPase
   (V-ATPase) as they mature from early endosomes. During maturation,
   pro-CTSD, an inactive propeptide, is cleaved into the mature enzyme in
   the acidic environment of lysosomes. Early Endosome Autoantigen-1
   (EEA1), lysosomal-associated membrane protein 1 (LAMP1), and V-ATPase D
   were used to identify early endosomes, lysosomes, and V-ATPase,
   respectively. IAAP treatment led to a significant increase in LAMP1,
   but no change in the expression of EEA1, mature CTSD, or V-ATPase D
   ([131]Figure 2F). Lyso-tracker confirmed that IAAP induced
   the accumulation of lysosomes ([132]Figure S2B). We estimated the
   lysosomal degradation function based on the degradation of the
   epidermal growth factor receptor (EGFR).[133]^39 Western blotting
   analysis showed that EGFR degradation was suppressed in IAAP-treated
   cells ([134]Figure 2G). Collectively, these results indicate that IAAP
   impairs lysosomal degradation activity without affecting lysosomal pH
   or fusion with autophagosomes, resulting in the excessive accumulation
   of autolysosomes.

IAAP interacts with CTSB and cathepsin L to suppress their bioactivity, of
which cathepsin L is critical for lysosomal function

   As shown previously ([135]Figures 2C–2G), IAAP inhibited lysosomal
   protein degradation. Based on the LC-MS/MS results, we speculated that
   IAAP directly interact with the lysosomal enzymes CTSB and CTSL. Using
   Co-IP, we further confirmed that IAAP interacts with the CTSB and CTSL
   precursors ([136]Figure 3A). IAAP inhibited CTSB and CTSL maturation in
   a time-dependent manner ([137]Figure 3B). Interestingly, the knockdown
   of CTSB increased the expression of CTSL in a feedback manner, and the
   protein levels of LC3B remained unchanged, whereas the level of SQSTM1
   decreased ([138]Figure 3C). However, the knockdown of CTSL resulted in
   a significant accumulation of the autophagy-related proteins LC3B and
   SQSTM1 ([139]Figure 3C). These results correspond to a study conducted
   by Xu et al.,[140]^40 suggesting that IAAP primarily affects lysosomal
   activity by inhibiting CTSL instead of CTSB.

Figure 3.

   [141]Figure 3
   [142]Open in a new tab

   IAAP interacts with CTSB and CTSL to suppress their bioactivity, of
   which CTSL is critical for lysosomal function

   (A) HBE cells were treated with biotin (10 μM), or IAAP-biotin (10 μM)
   for 24 h, the cell lysates were immunoprecipitated with
   beads-streptavidin and subjected to immunoblotting.

   (B) HBE cells were treated with IAAP for 6 h, and the levels of CTSL
   and CTSB were examined using Western blotting.

   (C) HBE cells were transfected with control-, CTSB- or CTSL-siRNA for
   24 h. The levels of LC3B, SQSTM1, LAMP1, CTSB, and CTSL were analyzed
   using Western blotting.

   (D) BMDM and THP-1 cells were treated with IAAP (5 μM) for 6 h, and HBE
   were treated with IAAP (5 μM) for 24 h. The CTSL activity in BMDM, HBE,
   and THP-1 cells was evaluated, n = 3 each group.

   (E) 3D schematic representation of IAAP bound to CTSL subsites. Best
   docking poses of IAAP at the active site of CTSL. The most relevant
   interacting residues were present in orange carbon polytubes, and
   ligands in blue carbon polytubes. DMSO was used as the vehicle control.
   All the quantitative data are presented as mean ± SEM and differences
   were identified using one-way ANOVA. ∗∗p < 0.01, ∗∗∗p < 0.001, and
   ∗∗∗∗p < 0.0001.

   We further examined the effect of IAAP on CTSL activity in BMDM, HBE,
   and THP-1 cells and validated the role of IAAP using a specific CTSL
   inhibitor, SID26681509.

   SID26681509 significantly suppressed CTSL activity but did not affect
   CTSS activity, suggesting its specificity ([143]Figures S3A–S3C). Our
   results showed that IAAP markedly inhibited CTSL activity, similar to
   that of SID26681509 ([144]Figure 3D). We observed that treatment with
   SID26681509 also led to autolysosome accumulation ([145]Figure S3D) and
   attenuated the degradation of EGFR as the concentration gradient
   increased ([146]Figure S3E), consistent with the effects of IAAP. Next,
   we confirmed by Co-IP that IAAP interacted with the precursor of CTSL
   stimulated with LPS ([147]Figure S3F). These results indicate that IAAP
   specifically binds to CTSL and suppresses its activity.

   Discovery Studio was used to calculate the affinity between CTSL and
   IAAP based on an efficient optimization algorithm for the scoring
   function. As shown in [148]Figures 3E and [149]S3G, the ligand could
   pass through the entrance of the enzyme and substrate cavity to replace
   the water molecules in the active sites and fit the narrow and large
   CTSL-binding pocket well, thereby generating inhibitory activity. The
   dimethylamino and azido groups located in the entrance cavity
   interacted with GLU 63 establishing an attractive force of charge. The
   1-methyl-indole fragment occupied the substrate cavity and formed
   pi-alkyl interactions with the lipophilic residues ALA 135, ALA 214,
   LEU 698, and MET 161. Moreover, CYS 25 and MET 70 established two
   Pi-sulfur interactions with the pyrimidine ring. In addition, the two
   amino groups on the pyrimidine formed a hydrogen bond interaction with
   ASP162. The above binding mode showed important interactions between
   IAAP and the surrounding residues. IAAP can bind to the active site of
   CTSL and contains strategically placed electrophilic warheads that trap
   the nucleophilic CYS 25 residue for activity. Collectively, these data
   indicate that IAAP targets and restrains lysosomal CTSL maturation,
   thereby inhibiting lysosomal degradation activity.

Cathepsin L is elevated in patients with acute respiratory distress syndrome
and in the murine model of lipopolysaccharide-induced acute lung injury

   To explore whether CTSL is associated with the modulation of ARDS, we
   first assessed the status of CTSL in ARDS by assessing CTSL activity in
   the BALF samples from patients with ARDS and healthy controls. CTSL
   activity significantly increased in the BALF of patients with ARDS
   ([150]Figure 4A). However, there was only a trend in Spearman’s rank
   correlation analysis of the relationship between CTSL activity and the
   levels of IL-6 but not statistically significant ([151]Figure 4B). In
   the murine model of LPS-induced ALI, CTSL activity was also enhanced in
   the BALF ([152]Figure 4C). Notably, CTSL activity was significantly
   correlated with the levels of the inflammatory cytokines IL-6 and CXCL1
   in the BALF ([153]Figure 4D). We also examined CTSS activity. The
   results showed that CTSS activity was increased in the BALF of mice
   with LPS-induced ALI which is consistent with previous
   research,[154]^41 but no increase was observed in the human BALF
   ([155]Figure S4). Western blot analysis showed that the CTSL protein
   level was significantly increased in the lung tissue ([156]Figure 4E).
   We further observed that LPS stimulation significantly increased the
   mature protein levels of CTSL without affecting lysosomal biosynthesis
   and acidification in vitro, whereas the protein level of LAMP1 declined
   ([157]Figure 4F). LPS stimulation enhanced lysosomal degradation
   activity ([158]Figure 4G). Western blot analysis demonstrated that the
   LPS-induced mature protein levels of CTSL were time-dependently
   suppressed by IAAP ([159]Figure 4H). These results suggested that an
   increase in mature CTSL may contribute to the development of ARDS.

Figure 4.

   [160]Figure 4
   [161]Open in a new tab

   CTSL is elevated in patients with ARDS and in the murine model of
   LPS-induced ALI

   (A) CTSL activity in the BALF from healthy controls (n = 4) and
   patients with ARDS (n = 6).

   (B) Spearman’s rank correlation analysis of the relationship between
   CTSL activity and the levels of IL-6 in the BALF from patients with
   ARDS (n = 6).

   (C) CTSL activity in the BALF from mice, n (NS) = 6, n (LPS) = 8.

   (D) Spearman’s rank correlation analysis of the relationship between
   CTSL activity and the levels of IL-6 or CXCL1 in the BALF from mice, n
   (NS) = 6, n (LPS) = 8.

   (E) CTSL protein levels in the lung tissue were examined using Western
   blotting, n (NS) = 4, n (LPS) = 5.

   (F) HBE cells were treated with 100 μg/mL LPS at different time points,
   and the levels of TFEB, EEA1, LAMP1, V-ATPase V1D, CTSD, and CTSL were
   examined using Western blotting.

   (G) Western blot analysis of EGFR after EGF incubation in cells with
   100 μg/mL LPS treatment at different time points.

   (H) HBE cells were treated with LPS (100 μg/mL) or IAAP (10 μM) at
   different time points, and the CTSL protein level was examined using
   Western blotting. All the quantitative data are presented as mean ±
   SEM. Differences between the two groups were identified using the
   Student’s t test. Correlations were analyzed using Spearman’s
   correlation analysis. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001; ns,
   p > 0.05.

Pharmacological inhibition of cathepsin L attenuates
lipopolysaccharide-induced inflammatory response and acute lung injury

   Next, we investigated whether CTSL is involved in the regulation of
   LPS-induced inflammatory responses using pharmacological inhibitors.
   The broad-spectrum cysteine protease inhibitor, E64D ([162]Figures S5A
   and S5C), and the CTSL-selective inhibitor, SID26681509, significantly
   reduced LPS-induced inflammation ([163]Figures 5A–5C), whereas the
   CTSB-selective inhibitor, CA074-ME, had little effect in vitro
   ([164]Figures S5B and S5D). In addition, both IAAP and SID26681509
   attenuated LPS-induced inflammatory response in BMDM from LC3B^−/- mice
   and LysM^Cre-ATG5^flox/flox mice ([165]Figures S5E and S5F). We
   investigated the therapeutic potential of CTSL-selective inhibitors in
   a murine model of LPS-induced ALI. SID26681509 treatment significantly
   alleviated LPS-induced lung inflammation ([166]Figures 5D–5F).
   Histopathological examination of lung sections revealed that
   SID26681509 significantly decreased inflammatory cellular infiltration
   ([167]Figure 5G), whereas SID26681509-treated mice survived longer than
   the control group after LPS administration ([168]Figure 5H). These
   results suggest that CTSL is a key regulator of the LPS-induced
   inflammatory response, and IAAP suppresses the LPS-induced inflammatory
   response through CTSL instead of autophagy.

Figure 5.

   [169]Figure 5
   [170]Open in a new tab

   Pharmacological inhibition of CTSL attenuates LPS-induced inflammatory
   response and ALI

   (A) BMDM were stimulated with LPS (100 ng/mL), or treated with
   SID26681509 (5 μM) for 6 h. Cells were then harvested to analyze
   secreted IL-6, CXCL1, and CXCL2 proteins in cell culture supernatants
   using ELISA, n = 3 each group.

   (B) HBE were stimulated with LPS (100 μg/mL) or treated with
   SID26681509 (1 μM) for 24 h. Cells were then harvested for analyzing
   secreted IL-6 and IL-8 proteins in cell culture supernatants using
   ELISA, n = 3 each group.

   (C) THP-1 were stimulated with LPS (100 ng/mL), or treated with
   SID26681509 (5 μM) for 6 h, Cells were then harvested for analyzing
   secreted IL-6, IL-8 proteins in cell culture supernatants using ELISA,
   n = 3 each group.

   (D) Mice were treated by intraperitoneal injection with SID26681509
   (20 mg/kg) 1h before pentobarbital injection and then with LPS (in
   50 μL saline) at a dose of 5 mg/kg or NS though intratracheal
   administration. Mice were sacrificed 24 h after LPS administration for
   analysis. Total and differential cell counts in the BALF from mice, n
   (DMSO) = 3, n (LPS) = 5, n (SID26681509) = 3, and n (SID26681509 +
   LPS) = 5.

   (E) Total protein concentration in the BALF.

   (F) Expression of IL6, CXCL1, and CXCL2 in the BALF.

   (G) Representative images of lung tissue with HE staining 24 h post-LPS
   challenge and histological inflammatory scores. Scale bar: 200μm.

   (H) For the survival assay, mice were treated intratracheally
   administered LPS (20 mg/kg) on day 0. Mice received an intraperitoneal
   injection of SID26681509 (20 mg/kg) or PBS 1 h before intratracheal
   instillation on day 0 and were injected with the drugs twice on day 3.
   Survival rates of mice after the intratracheal administration of LPS
   with or without IAAP intraperitoneal injection for 24 h. n (DMSO +
   LPS) = 17, n (SID26681509 + LPS) = 13. DMSO was used as the vehicle
   control. All the quantitative data are presented as mean ± SEM and
   differences were identified using one-way ANOVA. The log rank
   (Mantel-Cox) test was used to analyze survival rates. ∗p < 0.05,
   ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Conditional deletion of cathepsin L in myeloid cells significantly
ameliorated lipopolysaccharide-induced acute lung injury in mice

   To further explore the role of CTSL in ALI, myeloid cell-specific CTSL
   deletion mice (LysM^Cre-CTSL^flox/flox mice) were used. The deletion of
   CTSL in myeloid cells was achieved by crossing floxed CTSL mice
   (CTSL^flox/flox) with LysM^Cre mice ([171]Figure S6). In the
   LPS-induced ALI mouse model, conditional deletion of the myeloid cell
   CTSL significantly decreased the total cell count, neutrophil count,
   and protein concentration in the BALF ([172]Figures 6A and 6B). In
   addition, the levels of inflammatory factors in the BALF, including
   IL-6, CXCL1, and CXCL2, were markedly reduced in the BALF from
   LysM^Cre-CTSL^flox/flox mice compared to those in control mice
   ([173]Figure 6C). Consistently, mild inflammatory cell infiltration was
   observed in the lung tissues of LysM^Cre-CTSL^flox/flox mice
   ([174]Figure 6D), and the survival rate of LysM^Cre-CTSL^flox/flox mice
   was significantly increased after LPS exposure compared to that of
   control mice ([175]Figure 6E). These results suggested that CTSL
   facilitated LPS-induced inflammatory responses.

Figure 6.

   [176]Figure 6
   [177]Open in a new tab

   Conditional deletion of CTSL in myeloid cells significantly ameliorated
   LPS-induced ALI in mice

   (A) Mice were intratracheally treated with the LPS (in 50 μL saline) at
   a dose of 5 mg/kg or NS. Mice were then sacrificed 24 h after LPS
   administration for analysis. Total cell counts and differential cell
   counts in the BALF from mice, n (control-NS) = 4, n (control-LPS) = 4,
   n (LysM^Cre-CTSL^flox/flox-NS) = 4, n (LysM^Cre-CTSL^flox/flox -LPS) =
   5.

   (B) Total protein concentration in BALF.

   (C) Expression of IL6, CXCL1, and CXCL2 in the BALF.

   (D) Representative images of lung tissue with HE staining at 24 h post
   LPS challenge and histological inflammatory scores. Scale bar: 200 μm.

   (E) For surviving assay, mice were treated intratracheally with LPS
   (20 mg/kg) on day 0. Survival rates of mice after the intratracheal
   administration of LPS with or without IAAP intraperitoneally for 24 h.
   n (control -LPS) = 9, n (LysM^Cre-CTSL^flox/flox-LPS) = 8. All the
   quantitative data are presented as mean ± SEM and differences were
   identified using one-way ANOVA. The log rank (Mantel-Cox) test was used
   to analyzed survival rates. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001; ns,
   p > 0.05.

Cathepsin L is essential for lipopolysaccharide-induced K63-linked polyUb
binding of nuclear factor kappa B essential modulator by degrading A20

   After clarifying the protective effect of the CTSL inhibitors on the
   LPS-induced inflammatory response, we further explored the regulatory
   mechanism of CTSL. Given that the NF-κB signal pathway was the key
   mechanism to mediate LPS-induced inflammation, we examined whether
   CTSL-selective inhibitors affect the NF-κB signal pathway. CTSL
   inhibitors concentration-dependently reduced the phosphorylation of
   IκBα and p65, whereas it was quite unexpected that CTSL inhibitors led
   to a significant accumulation of the phosphorylated IKKα/β in BMDM and
   HBE cells ([178]Figures 7A, [179]S7A, and S7B). The IκB kinase complex
   (IKK) consists of IKKα, IKKβ, and NEMO. In response to stimulation by
   LPS, K63-linked polyUb recruits the IKK complex by binding to NEMO, and
   then IKK is activated to phosphorylate IκBα, leading to NF-κB
   activation.[180]^18^,[181]^19^,[182]^42 The proximity ligation assay
   (PLA) showed strong signals for interactions between NEMO and
   K63-linked polyUb upon LPS administration, whereas CTSL inhibitors
   reduced their associations ([183]Figure 7B), suggesting that the
   ubiquitination of NEMO was modulated by CTSL in the context of
   LPS-induced inflammatory responses.

Figure 7.

   [184]Figure 7
   [185]Open in a new tab

   CTSL regulates the LPS-induced K63-linked polyUb binding of NEMO by
   degrading A20

   (A) BMDM cells were treated with IAAP or SID26681509 for 6 h, then
   stimulated with LPS (100 ng/mL) for 5 min, the levels of p-IKKα/β,
   IκBα, p-IκBα, p65, p-p65, and ACTB were examined using Western
   blotting.

   (B) BMDM cells were treated with IAAP or SID26681509 for 6 h, then
   stimulated with LPS (100 ng/mL) for 5 min. BMDM was analyzed for the
   spatial approximation of IKKγ with K63-linked polyUb components using
   PLA. Red, proximity ligation-positive signals. Scale bars: 5 μm. The
   quantification shown on the right represents the fluorescence, n = 15.

   (C) BMDM cells were treated with IAAP or SID26681509 for 6 h, then
   stimulated with LPS (100 ng/mL) for 5 min, the levels of A20, NEMO,
   IKKβ, and ACTB were examined using Western blotting.

   (D) BMDM cells were analyzed for the spatial approximation of A20 with
   CTSL components using PLA. Red, proximity ligation-positive signals.
   Scale bars: 5 μm. Quantification shown on the right represents the
   fluorescence, n = 15.

   (E) BMDM cells were transfected with control-, A20-siRNA for 24 h. Then
   cells were treated with IAAP (5 μM) or SID26681509 (5 μM) for 6 h,
   and then stimulated with LPS (100 ng/mL) for 5 min. Cells were then
   harvested for analyzing the mRNA expression of IL-6 and CXCL1 using
   qRT-PCR, n = 3 in each group. DMSO was used as the vehicle control. All
   the quantitative data are presented as mean ± SEM and differences were
   identified using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001,
   and ∗∗∗∗p < 0.0001; ns, p > 0.05.

   A20 is a deubiquitinating enzyme that removes K63-linked polyUb from
   the target protein and thereby negatively regulates
   NF-κB.[186]^42^,[187]^43 Consistent with previous reports, the PLA
   indicated that LPS increased the colocalization of A20 and LAMP1,
   suggesting that lysosomes are the primary A20 degradation pathway
   ([188]Figure 7D).[189]^44^,[190]^45 We found that LPS treatment led to
   a decrease in A20 levels, which was significantly reversed by the CTSL
   inhibitors ([191]Figures 7C, [192]S7A, and S7B). Similar results were
   observed that genetic knockout of CTSL effectively reduced the protein
   levels of p-IκBα and p-p65, but rescued A20 degradation in response to
   LPS, and elevated p-IKKα/β protein ([193]Figures S7C). Thus, we
   hypothesized that CTSL directly degrades A20 in lysosomes. As expected,
   we observed that the association between CTSL and A20 increased after
   LPS treatment, and was significantly reduced by CTSL inhibitors
   ([194]Figure 7D). We also showed that LPS stimulation promoted the
   binding of A20 to CTSL ([195]Figure S7D), and CTSL inhibitors barely
   suppressed LPS-induced inflammation when A20 was knocked down,
   suggesting that CTSL-mediated LPS-induced inflammatory responses are
   dependent on A20 ([196]Figure 7E). These experiments collectively
   indicated that the LPS-induced upregulation of CTSL targeted to degrade
   A20 in lysosomes and regulated the K63-linked polyUb binding of NEMO,
   thereby activating the NF-κB pathway.

Discussion

   In this study, we describe IAAP as a potent small-molecule inhibitor
   that targets the degradation activity of the lysosomal enzyme CTSL. We
   determined that CTSL is involved in the regulation of LPS-induced NF-κB
   activation and pulmonary inflammation. Mechanistically, LPS triggers
   the maturation of CTSL and enhances its lysosomal activity, leading to
   the degradation of A20, and results in a significant accumulation of
   K63-polyubiquitinated NEMO, promoting the kinase activity of the IKK
   complex and subsequently NF-κB activation. Pharmacological inhibition
   and genetic knockout of CTSL rescued LPS-induced degradation of A20
   reduced K63-polyubiquitinated NEMO and eventually attenuated
   LPS-induced inflammatory responses.

   ALI/ARDS is an acute inflammatory disease associated with high
   morbidity and mortality rates. Curative therapy for ALI/ARDS remains a
   significant challenge in critical care medicine and no specific
   pharmacotherapy has been proven to be effective. We synthesized the
   small-molecule compound IAAP as a candidate anti-inflammatory
   drug.[197]^38 Our current study showed that IAAP ameliorates
   LPS-induced inflammatory responses in vitro and ALI in vivo. Using
   LC-MS/MS and KEGG pathway enrichment analysis, we showed that IAAP
   impaired the autophagy-lysosome pathway by reducing the mature protein
   levels of lysosomal cathepsins, CTSB, and CTSL. Knockdown of CTSB
   enhanced CTSL expression, and CTSL overexpression promoted
   autophagosome degradation as reported previously,[198]^46 suggesting
   that IAAP exerts its effect through CTSL instead of through CTSB. Our
   previous study validated that LPS inhibits autophagy by activating
   mTORC1, which then activates the NF-κB signaling to promote
   inflammation in ALI.[199]^16 We next determined that both IAAP and
   SID26681509 attenuated LPS-induced inflammatory response in BMDM from
   Lc3b-deficient and LysM^Cre-Atg5^flox/flox mice. Thus, our results
   suggest that IAAP regulates the LPS-induced inflammatory response
   through CTSL instead of autophagy and that CTSL may play a critical
   role in the regulation of the LPS-induced inflammatory response.

   The lysosome serves not only as the center for degradation and
   metabolism but also as a hub for several signaling pathways. Most
   lysosomal studies have focused on metabolic disorders,
   neurodegenerative diseases, and tumors.[200]^21^,[201]^47^,[202]^48 In
   recent years, lysosomal enzymes, particularly cathepsins, have received
   increasing attention as potential targets for the treatment of
   inflammatory diseases. LPS or IFN-γ increases cathepsins expression in
   macrophages.[203]^49^,[204]^50 CTSB, CTSL, and CTSS are overexpressed
   in lung tissues of patients with cystic fibrosis.[205]^51^,[206]^52
   CTSB could enter the cytoplasm and activate NLRP3 inflammasomes,
   leading to the maturation and secretion of cytokines such as IL-1β,
   IL-6, and TNFα.[207]^53 In patients with ALI/ARDS, McKelvey et al. have
   reported that CTSS is elevated in the lungs of patients with ALI/ARDS
   and in animal models of ALI/ARDS. CTSS may partially mediate its
   pathogenic effects via protease-activated receptor-1.[208]^41 This
   study investigated the status and role of CTSL in patients with ALI and
   ARDS. CTSL levels were elevated in the BALF samples from patients with
   ARDS and in a murine model of ALI. LPS increased the mature protein
   levels of CTSL without affecting lysosomal biosynthesis, acidification,
   or maturation. It is worth emphasizing that the maturation of CTSL was
   associated with LPS-induced inflammation. Using the pharmacological
   inhibition of cathepsins and myeloid cell-specific CTSL deletion in
   mice (LysM^Cre-CTSL^flox/flox mice), we confirmed that CTSL plays a
   critical role in the regulation of LPS-induced inflammation.

   In addition, we examined the effects of CTSL-siRNA on LPS-induced
   inflammation in vitro and determined that the knockdown of CTSL barely
   affected the LPS-induced inflammatory response (data not shown). One
   plausible explanation is that CTSL is a dual-function molecule, whose
   functional switch is regulated by its maturation state similar to
   CTSD.[209]^54 Pro-CTSL may have important consequences for normal
   cellular physiological processes, whereas CTSL-siRNA reduced both
   pro-CTSL and mature-CTSL protein levels instead of inhibiting their
   enzymatic activity as CTSL inhibitors do.

   The canonical NF-κB pathway responds to diverse immune stimuli such as
   LPS and TNFα, resulting in sharp but transient NF-κB
   activation.[210]^55 Yang et al. have reported that the increased CTSB
   decreased TNFα-mediated NF-κB activation in HDAC3-deficient macrophages
   as a scissor for the significant lysosomal degradation of RIP1.[211]^56
   Similarly, we showed that CTSL plays a critical role in regulating
   NF-κB activation and function but has a mechanism distinct from that of
   CTSB. In the canonical NF-κB pathway, IκBα is a negative regulator
   downstream of the NF-κB signaling. Upon activation, IκBα is
   phosphorylated by the IKK complex, after which IκBα is degraded,
   leading to the translocation of NF-κB into the nucleus. There, it
   activates the expression of numerous genes, including those involved in
   immune and inflammatory responses. The IKK complex consists of IKKα,
   IKKβ, and NEMO. Phosphorylated IKKβ and K63-polyubiquitinated NEMO are
   required for the kinase function.[212]^18^,[213]^19 A20 is an important
   ubiquitin editing enzyme involved in deubiquitination modifications
   such as K63-linked polyUb in NF-κB signaling.[214]^57^,[215]^58
   Following LPS stimulation, A20 is bound by lysosomal-associated protein
   transmembrane 5 (LAPTM5) and transported to the lysosome for
   degradation, thereby activating the NF-κB signaling.[216]^44 Notably,
   our study determined that the LPS-induced up-regulation of CTSL is
   targeted to degrade A20 in the lysosomes and decrease K63-linked polyUb
   binding of NEMO to maintain the IKK complex activation. In addition, we
   showed here that the recruitment of the IKK complex and phosphorylation
   of IKKβ could happen independent of the K63-linked polyUb binding of
   NEMO. CTSL-selective inhibitor effectively leads to the accumulation of
   phosphorylated IKKβ, however, a reduction of K63-linked polyUb binding
   of NEMO, thereby inhibiting the activation of NF-κB signaling and the
   LPS-induced inflammation and ALI.

   In summary, the present study showed that IAAP targets lysosomal CTSL
   activity to suppress the LPS-induced ALI through deubiquitinase A20 to
   modulate the NF-κB signaling. Our findings suggest that CTSL is a
   valuable therapeutic target and IAAP can serve as a potential lead
   compound for the development of new drugs for ALI.

Limitations of the study

   This study has several limitations. First, although we have utilized
   the most reliable research methods available to assess CTSL activity,
   there is some controversy regarding the substrate used for detecting
   cathepsin activity. This is a problem within the field that requires
   further in-depth study. Second, the precursor and mature forms of CTSL
   may exert differential effects on cellular functions, including the
   inflammatory response, which warrant further investigation.

Resource availability

Lead contact

   Further information and requests for resources and reagents should be
   directed to and will be fulfilled by the lead contact, Zhihua Chen
   (Email: zhihuachen@zju.edu.cn).

Material availability

   This study did not generate new, unique reagents.

Data and code availability

     * •
       The supplemental information contains most results. Additional
       results, including data going into all figures and tables, are
       available from the [217]lead contact.
     * •
       This article does not report the original code.
     * •
       Any additional information needed to reanalyze the data reported in
       this study may be requested from the [218]lead contact.

Acknowledgments