Blister Fluid Induces MMP-9-Associated M2-Type Macrophages in Bullous Pemphigoid.

Bullous pemphigoid (BP) is a cutaneous autoimmune disease, characterized by an inflammatory cascade leading to blister formation. Although macrophages were shown to participate in BP pathophysiology, their role in the blister formation process still needs to be investigated. We here addressed the influence of serum and blister fluid (BF) from patients with BP on the polarization status of macrophages with regards to the metalloproteinase-9 (MMP-9) expression. We demonstrated that several markers related to the alternatively activated macrophage phenotype (M2) including IL-10, TARC, arginase, TNFα, and IL-1RA were meaningfully increased in BF of patients with BP. We further showed that BF, but not serum from patients with BP, significantly induced the expression of CD163, CD206, and IL-10 in BP monocyte-derived macrophages (MDMs). Notably IL-10 was the only cytokine to be correlated to the reference clinical score, BP disease activity index (BPDAI), especially to the inflammatory BPDAI subscore evaluating urticarial and erythematous skin lesions (r = 0.57, p = 0.0004). We also found elevated levels of MMP-9 to M2-type macrophages ex vivo and highlighted the presence of CD163+ MMP-9+ macrophages histologically, at skin lesional site. Finally, we showed that methylprednisolone reduced MMP-9 levels in MDMs without modifying the other M2 markers. All together these results strongly support the presence of M2-phenotype macrophages with pro-inflammatory properties susceptible to favor blister formation in BP.

Bullous pemphigoid outcome is associated with CXCL10-induced matrix metalloproteinase 9 secretion from monocytes and neutrophils but not lymphocytes.

BACKGROUND: The outcome of bullous pemphigoid (BP), the most frequent autoimmune skin-blistering disease, involves matrix metalloproteinase 9 (MMP-9), IL-17, and IL-23 release from infiltrated inflammatory cells. The chemokine CXCL10 has been associated with several autoimmune diseases, but its participation in BP pathophysiology still needs to be clarified. OBJECTIVE: We sought to assess whether BP outcome was associated with different CXCL10 levels and to evaluate the contribution of CXCL10 to the described cytokine/protease inflammatory loop associated with disease outcome. METHODS: Skin biopsy specimens (n = 16), serum (n = 114), blister fluid (n = 23), and primary inflammatory cells from patients with BP were used to investigate CXCL10 expression and function. RESULTS: At baseline, both resident cells, such as keratinocytes and fibroblasts, and infiltrating immune cells expressed CXCL10 at lesional sites in skin of patients with BP. CXCL10 levels were higher in blister fluid (P < .0001) and serum (P < .005) from patients with BP than in serum from age- and sex-matched control subjects (n = 34). Furthermore, CXCL10 serum levels increased at day 60 only in patients who relapsed within the first year of treatment (n = 33, P < .005). Interestingly, CXCL10 expression could be upregulated by itself and IL-17 in inflammatory cells. Notably, neutrophils and monocytes from patients with BP, but not lymphocytes, responded to CXCL10 by increasing MMP-9 secretion through the activation of extracellular signal-regulated kinase 1/2, p38, phosphoinositide-3 kinase signaling pathways. Finally, CXCL10-increased MMP-9 secretion was inhibited by methylprednisolone and also by compound A, a novel nonsteroidal glucocorticoid receptor ligand. CONCLUSION: We showed that increased levels of inflammatory biomarkers in patients with BP, such as CXCL10, favor neutrophil- and monocyte-associated MMP-9 release and disease relapse and opened new therapeutic horizons in patients with this autoimmune disease.

Abnormal recruitment of extracellular matrix proteins by excess Notch3 ECD: a new pathomechanism in CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the NOTCH3 receptor (Notch3(ECD)) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3(ECD)-containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel pathology. Herein, we hypothesized that excess Notch3(ECD) could recruit and sequester functionally important proteins within small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immunohistochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and artery samples demonstrated that mutant Notch3(ECD) accumulates in disulphide cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3(ECD)-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the formation of Notch3(ECD)-TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of both insoluble cross-linked and soluble TIMP3 species. Moreover, reverse zymography assays show a significant elevation of TIMP3 activity in the brain vessels from mice and patients with CADASIL. Collectively, our findings lend support to a Notch3(ECD) cascade hypothesis in CADASIL disease pathology, which posits that aggregation/accumulation of Notch3(ECD) in the brain vessels is a central event, promoting the abnormal recruitment of functionally important extracellular matrix proteins that may ultimately cause multifactorial toxicity. Specifically, our results suggest a dysregulation of TIMP3 activity, which could contribute to mutant Notch3(ECD) toxicity by impairing extracellular matrix homeostasis in small vessels.