key: cord-0959936-0qz0s5z1 authors: Mohammadhosayni, Mina; Sadat Mohammadi, Fatemeh; Ezzatifar, Fatemeh; Mahdavi Gorabi, Armita; Khosrojerdi, Arezou; Aslani, Saeed; Hemmatzadeh, Maryam; Yazdani, Shahrooz; Arabi, Mohsen; Marofi, Faroogh; Jadidi-Niaragh, Farhad; Shomali, Navid; Mohammadi, Hamed title: Matrix metalloproteinases are involved in the development of neurological complications in patients with Coronavirus disease 2019 date: 2021-08-17 journal: Int Immunopharmacol DOI: 10.1016/j.intimp.2021.108076 sha: 2a839afda546ece76b1f0c6348c2bc962c3e329b doc_id: 959936 cord_uid: 0qz0s5z1 BACKGROUND: Evidence show that Matrix metalloproteinases (MMPs) have been associated with neurological complications in the viral infections. Here in the current investigation, we intended to reveal if MMPs are potentially involved in the development of neurological symptoms in the patients with Coronavirus disease 2019 (COVID-19). METHODS: The levels of MMPs, inflammatory cytokines, chemokines, and adhesion molecules were evaluated in the serum and cerebrospinal fluid (CSF) samples from 10 COVID-19 patients with neurological syndrome (NS) and 10 COVID-19 patients lacking NS. Monocytes from the CSF samples were treated with TNF-α and the secreted levels of MMPs were determined. RESULTS: The frequency of monocytes were increased in the CSF samples of COVID-19 patients with NS compared to patients without NS. Levels of inflammatory cytokines IL-1β, IL-6, and TNF-α, chemokines CCL2, CCL3, CCL4, CCL7, CCL12, CXCL8, and CX3CL1, MMPs MMP-2, MMP-3, MMP-9, and MMP-12, and adhesion molecules ICAM-1, VCAM-1, and E-selectin were significantly increased in the CSF samples of COVID-19 patients with NS compared with patients without NS. Treatment of CSF-derived monocytes obtained from COVID-19 patients with NS caused increased production of MMP-2, MMP-3, MMP-9, and MMP-12. CONCLUSIONS: Higher levels of inflammatory cytokines might promote the expression of adhesion molecules on blood-CSF barrier (BCSFB), resulting in facilitation of monocyte recruitment. Increased levels of CSF chemokines might also help to the trafficking of monocytes to CSF. Inflammatory cytokines might enhance production of MMPs from monocytes, leading to disruption of BCSFB (and therefore further infiltration of inflammatory cells to CSF) in COVID-19 patients with NS. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that cause coronavirus disease 2019 (COVID -19) has been associated with a wide range of clinical symptoms that is presented as acute respiratory distress syndrome (ARDS) in the severe forms. The virus targets and attacks various body organs where angiotensin-converting enzyme 2 (ACE2) receptor is exerted as the major receptor for virus S protein [1, 2] . Among the typical clinical manifestations of COVID-19 are fever, fatigue, dry cough, and sore throat [3] . A growing body of evidence reveals the neurological symptoms in COVID-19 subjects such as headache, neuroinflammatory presentations, and cerebrovascular complications [4] . Molecular tests have recognized the SARS-CoV-2 nucleic acid in the cerebrospinal fluid (CSF) of COVID-19 patients [5] . Moreover, virus particles have also been detected in the autopsy samples of brain in a subject [6] . Matrix metalloproteinases (MMPs) are zinc-dependent enzymes that degrade extracellular matrix (ECM) proteins, such as collagen, fibronectin, and laminin as well as basement membrane structures. MMPs have been associated with diverse pathophysiological conditions like inflammation as well as metastasis and angiogenesis in malignancies [6, 7] . These enzymes play a role in promoting the passing of inflammatory immune cells through the blood-brain barrier (BBB) and accumulation in the CSF and Central nervous system (CNS) [8] . Serum MMP-3 concentration was reported to be higher in COVID-19 patients and correlated with increased levels of inflammatory mediators [9] . Increased levels of chemokines as well as MMPs, like MMP-2, MMP-3, MMP-8, and MMP-9 in the CSF of patients with Varicella-zoster virus (VZV) infection were reported [7] . Increased expression levels of MMP-3, MMP-12, and tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) was reported in mice with lethal infection with neurotropic mouse hepatitis virus compared to sublethal infection. Moreover, activity of MMP-9 was associated with neutrophil recruitment to CNS [8] . In the CSF of patients with human immunodeficiency virus (HIV) dementia, increased levels of pro-MMP-2 and pro-MMP-7 were detected. Even though high serum levels of MMPs might be associated with promoted levels of MMPs in the CSF, the brainderived cells produced MMP-2, MMP-7, and MMP-9, which were stimulated upon induction of these cells with tumor necrosis factor (TNF)-α. Hence, increased levels of MMPs in the CSF mirrored the aberrant/hyper activation of immune system in CNS of patients with HIV dementia [9] . CNS is regarded as an immune-privileged site and, therefore, trafficking of immune cells into the CNS is limited through the blood-brain barrier (BBB) as well as the blood-CSF barrier (BCSFB). That notwithstanding, immune surveillance occurs steadily in the CNS during normal physiological conditions, demonstrating that infiltration of immune cells into CNS happens regardless of inflammation or BBB/BCSFB injury [10, 11] . On the other hand, pathological conditions like inflammation promote the infiltration of immune cells into CNS that is occurred when BBB is injured and is mediated by chemokines. In such condition, immune cells present in the parenchymal basement membrane surrounding the spinal cord and brain enters the CNS [12] . MMPs and adhesion molecules have been hypothesized to be involved in the CNS injury during coronavirus infections. After entry of coronaviruses into lung, they infect epithelial cells and may pass across the epithelium and enter into blood to infect the monocytes. MMPs, especially MMP-9 (induces the permeability of BBB) and inflammatory mediators, like TNF-α (that triggers overexpression of Intercellular adhesion molecule 1 (ICAM-1) on the endothelial cells) contribute the infected immune cells (like monocytes) to pass through the BBB and enter into CNS. These infected immune cells might produce inflammatory mediators in the CNS and cause neuron injury. These infected immune cells also secret several chemokines, such as CCL5, CXCL10, CXCL11, leading to infiltration of inflammatory immune cells (like T cells) into CNS [10] . The major mechanobiology underlying neurologic syndrome (NS) and neurovirulence by SARS-CoV-2 has not been clarified yet. Here in this study, we tried to explore possible mechanism by investigating the levels of different chemokines, MMPs, as we as adhesion molecules in COVID-19 patients. In this study, 10 subjects with COVID-19 presenting neurological manifestations and 10 age-and sex-matched COVID-19 patients without neurological presentations were included (Table 1) CSF samples and serum samples isolated from peripheral blood of all study subjects were evaluated using enzyme linked immunosorbent assay (ELISA) to measure the concentration of Total leukocytes were isolated from the CSF samples by centrifugation. Additionally, peripheral blood mononuclear cells (PBMCs) were isolated from blood samples after dilution in Phosphatebuffered saline (PBS) by density-gradient centrifuged using Ficoll/Hypaque 1.077 g/ml The Mann-Whitney U-test was used to compare the data between the groups. Numeric and nominal data presentation was performed by mean ± standard deviation (SD) and numbers and percentage, respectively. Analysis of data and designing of graphs were conducted by GraphPad PRISM software v.8.00 (GraphPad Software, Inc., San Diego, CA, USA). Demographic data and clinical presentations of study subjects are listed in Table 1 . Study subjects were composed of 10 cases with NS and 10 individuals without NS. In both groups, 5 (50%) male cases and 5 (50%) female subjects were included. Among the laboratory tests, both groups had similar levels of white blood cell (WBC), lymphocyte-total leukocyte ratio, neutrophil-lymphocyte ratio, C-reactive protein (CRP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH). It was observed that the SARS-CoV-2 nucleic acid PCR of peripheral blood was positive in 4 (40%) subjects with NS and in 1 (10%) patient without NS. The results of SARS-CoV-2 nucleic acid PCR was negative in all patients. The time from COVID-19 initial diagnosis to lumbar puncture to obtain CSF samples was 6.2±8.7 days in subjects with NS and 5.5 ± 7.9 days in subjects without NS. The neurological presentations of the 10 COVID-19 subjects with NS are shown in Table 2 . The frequency of leukocytes in the CSF of subjects with NS was 10.12±4.40 cell/μl while it was less than 1 cell/μl in patients without NS (P< 0.01; Figure 1 .A). Most importantly, it was observed that the number of monocytes in the CSF samples from COVID-19 cases with NS was significantly higher compared to that of cases without NS (2.55±0.54 cell/μl vs. 0.018±0.006 cell/μl; P<0.001; Figure 1 .B). The Experiments revealed that the levels of IL-1β had no significant differences in both serum and CSF samples from COVID-19 cases with NS in comparison to COVID-19 cases without NS ( Figure 3 .A). Although IL-6 level had no significant difference in the serum samples of COVID-19 cases with NS compared to patients without NS, it was significantly higher in the CSF samples of COVID-19 patients with NS (P= 0.0004; Figure 3 .B). Additionally, the TNF-α level was significantly higher in the CSF sample, but not serum sample, of COVID-19 patients with NS compare to patients without NS (P= 0.0007; Figure 3 .C). The levels of CCL2, CCL3, CCL4, CCL7, CCL12, CXCL8, and CX3CL1 was higher in both serum and CSF samples from COVID-19 cases with NS compared to patients without NS. Nonetheless, no significant differences were detected in serum and CSF CCL-5 levels between COVID-19 patients with and without NS ( Figure 4 ). It was detected that ICAM-1 level was significantly higher in the serum (P= 0.0005) and CSF (P= In the supernatant of TNF-α treated monocytes derived from CSF samples of COVID-19 patients with NS, levels of MMP-2 (P= 0.0002; Figure 6 .A), MMP-3 (P= 0.0007; Figure 6 .B), MMP-9 (P= 0.0074; Figure 6 .E), and MMP-12 (P= 0.0056; Figure 6 .F) was significantly higher in comparison to untreated monocytes obtained from CSF samples of COVID-19 patients with NS. However, no significant differences were observed in secreted levels of MMP-7 (Figure 6 .C) and MMP-8 ( Figure 6 .D). Additionally, we performed the same experiment in the monocytes obtained from CSF of COVID-19 cases without NS, but there were no statistically significant differences between levels of MMPs before and after treatment of monocytes with TNF-α (Data not shown). Research show that numerous viruses are able to invade the CNS and infect the immune cells as well as neurons. In spite of little understandings with respect to invasion of viruses into CNS, such events are not rare and are progressively identified through advancements in sophisticated diagnostic tools. Viruses exert various routs to enter CNS and infect glial cells as well as neurons and contribute to the development of neurological disorders [13] . Evidence shows that patients with COVID-19 suffer from several forms of neurological complications [14] . Approximately 20% of subjects with COVID-19 who need ICU admission have shown neurological manifestations that are probably at increased susceptibility to mortality [15] . Infection by SARS-CoV-2 was associated with postmortem neuropathological modifications. In addition, the virus was identified in the brain of COVID-19 cases with preexisting microvascular disorders that exhibited encephalopathy. As a result, microvascular comorbidities might confer an increased susceptibility towards neurological disorders in patients with COVID-19 [16] . ACE2 are expressed on the endothelium of various tissues, including brain [17] . It was reported that SARS-CoV-1 were able to bind directly to the ACE2 expressed on the endothelial cells of the cerebral microvasculature. Therefore, it seems that SARS-CoV-2 might also be able to bind the BBB endothelial cells, resulting in accesses of virus to CNS and infect neurons and glial cells [18, 19] . of the brain ventricles [20, 21] . The gene expression profile of choroid plexus might be modulated through the inflammation caused by coronaviruses, leading to disrupted integrity of BCSFB [22] . Additionally, such inflammation might activate nuclear factor (NF)-κB pathway, leading to overproduction of MMP-9 and increased permeability of BCSFB [23] . These events might also upmodulate the expression of CCL-2, IL-1, IL-6, TNF-α, MMP-8, and ICAM-1 that further promote the permeability of BCSFB as well as infiltration of inflammatory immune cells [24] [25] [26] . Moreover, higher levels of pro-MMP-2 and pro-MMP-7 were detected in the CSF of patients with HIV dementia. The brain-derived cells produced MMP-2, MMP-7, and MMP-9, which were triggered after induction of these cells with TNF-α. Therefore, increased levels of MMPs in the CSF demonstrate the aberrant/hyper activation of immune system in CNS of patients with HIV dementia [9] . In the Japanese encephalitis virus infected mice, increased expression and activity of MMP-2 and MMP-9 in the serum samples [27] . The human coronavirus OC43 strain was observed to modulated MMP-2 and MMP-9 activity produced by the astrocytic and microglial cell lines [28] . Therefore, viruses might develop neuropathology through promoting Previously, it was seen that MMP-3 concentration was higher in serum of COVID-19 patients that correlated with increased levels of inflammatory cytokines [9] . In addition, inflammatory cytokines have been shown to promote the production of MMPs from monocytes [29] . 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This study was supported by a grant from Alborz University of Medical Sciences (Grant No. 99-4209).