{'fraction_non_alphanumeric': 0.040771812080536915, 'fraction_numerical': 0.03557046979865772, 'mean_word_length': 4.839647289353364, 'pattern_counts': {'":': 0, '<': 20, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
CCR2 and DPP9 expression in the peripheral blood of COVID-19 patients: Influences of the disease severity and gender

Graphical abstract


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)related COVID-19 seriously affected more than 212 territories and countries (Cucinotta and Vanelli, 2020;. Since the SARS-CoV-2 receptor (ACE2: angiotensin-converting enzyme 2) is expressed by different types of cells and tissues, thus various organs, in particular lungs, intestine, testis, heart, kidneys, esophagus, bladder, brain and liver are invaded by SARS-CoV2 . COVID-19-related clinical manifestations emerge following a latent time of about 5 days with a range of 2-14 days . Based on the World Health Organization (WHO) criteria, SARS-CoV-2mediated COVID-19 is categorized into several patterns, including mild, moderate, severe, and critical patterns (Organization, 2021). Patients with COVID-19 mostly exhibit mild forms of COVID-19, while, some patients progress to the moderate, severe, and critical forms, which may express several organ dysfunctions, in particular acute respiratory distress syndrome (ARDS) (Mitra et al., 2020).
Large number of the inflammatory cells (such as monocytes, macrophages, lymphocytes and neutrophils) infiltrate the respiratory system during COVID-19 . Cytokine storm and hyper inflammatory responses perform a crucial role in the Development of COVID-19-related complications Jafarzadeh et al., 2020). Among the cytokine storm-related factors, chemokines guide the leukocyte infiltration into the inflamed tissues (Jafarzadeh et al., 2020;Jafarzadeh et al., 2019). CC chemokine receptor 2 (CCR2) is mainly expressed by certain cell types, such as monocytes, dendritic cells (DCs), macrophages, NK and T lymphocytes, although it can be induced by other cells under inflammatory situations (Fantuzzi et al., 2019;Wareing et al., 2007). C-C motif chemokine ligand 2 (CCL2), CCL7, CCL8, CCL13, and CCL16 are known as the CCR2 ligands, but CCL2 has been considered as the most potent ligand (Fantuzzi et al., 2019;Palomino and Marti, 2015). Monocytes/macrophages are the major producers of CCL2 (Fantuzzi et al., 2019). MCP-1 interaction with CCR2 play a key role in the induction of inflammation and inflammation-related diseases. Moreover, CCR2 binding by MCP-1 support innate immunity response by recruiting monocytes into inflammatory sites (Bianconi et al., 2018). CCL2/CCR2 pathway is a key player in the trafficking of lymphocytes and monocytes/macrophages have been implicated in the pathogenesis of various diseases, such as viral infections (Fantuzzi et al., 2019). In the infectious diseases process, the expression of CCR2 in peripheral B and T cells strongly elevates and suggests the crucial role of CCR2 in immunomodulation (Hodge et al., 2012). Moreover, it is indicated that by inhibition the pathway of CCR2 interaction in immunity defence in influenza models the levels of cytokine storm mediators including interleukin-6, tumor necrosis factor-α, interferon-γ, and macrophage inflammatory protein 2 was decreased dramatically (Dessing et al., 2007). Such findings highlight the importance of CCR2 in viral diseases such as COVID-19.
Inflammasomes as the amplifiers of the inflammation are also activated by various types of endogenous and exogenous components. Inflammasome activation results in pro-IL-1β and pro-IL-18 maturation into their active forms, and pyroptosis enabling the release of IL-1β and IL-18 which recruit neutrophils, monocytes, and macrophages to the infection sites. Dipeptidyl peptidase 9 (DPP9) blocks the C terminus of NLR Family Pyrin Domain Containing 1 (NLRP1) to prevent inflammasome activation (Hollingsworth et al., 2021). Additionally, NLR Family Pyrin Domain Containing 3 (NLRP3) activates the DPP9 that leads to inflammasome inhibition (Okondo et al., 2017;Okondo et al., 2018;Zhong et al., 2018). DPP9 is extensively expressed in lymphocytes and epithelial cells, but it is up-regulated upon activation of T or B cells (Yu et al., 2009;Abbott et al., 2000;Chowdhury et al., 2013). Although DPP9 is located intracellularly; it is suggested that under certain conditions it may also be translocated on the surface of immune cells (Bank et al., 2011). DPP9 also can act as a MERS-coronavirus receptor (Raj et al., 2013). In addition, it is demonstrated that DPP enzymes family such as DPP9 are crucial immunomodulatory factors, hence, Tlymphocyte proliferation and pro-inflammatory cytokine production could be decreased through reducing the expression or by inhibition of these enzymes (Reinhold et al., 2009;Lankas et al., 2005). Also, it is previously shown that DPP8/9 inactivation leads to T-cell repression and IL-2 release in human in vitro models (Huang et al., 2021). Therefore, these observations emphasize the importance of these enzymes and their role in the immune system as factors that have received less attention in infectious diseases.
Considering the fundamental role of CCR2 and DDP9 in the induction and regulation of the inflammatory responses, this study aimed to evaluate the expression levels of CCR2 and DDP9 in the peripheral blood cells collected from COVID-19 patients, in an attempt to explore the possible association of their expression levels with disease severity and gender of patients.

Subjects
Totally, from 1 to 10 July 2021, 470 subjects (235 male and 235 female; aged 20-80 years) with quantitive real-time PCR (RT-qPCR)confirmed COVID-19 who were referred to Imam Khomeini hospital of Jiroft (a city located in the southeast of Iran) were enrolled to this casecontrol study. The patients who had a history of other inflammatory diseases, negative RT-qPCR, or personal dissatisfaction were excluded from the study. According to the WHO guidance concerning the COVID-19-related clinical symptoms and management, the patients were divided into three subgroups including moderate, severe, and critical patients (Table 1). In addition, 100 healthy individuals (50 male and 50 female) without any inflammatory diseases were selected as the control group. A peripheral blood sample was collected from participants for RNA extraction. The Ethics Committee of Kerman University of Medical Sciences evaluated and approved the research protocol that was registered with code: IR.KMU.REC.1400.411.

RNA extraction and real time-PCR
Commercial kits (Qiagen, Germany) were used to extract total RNA from peripheral blood samples according to the manufacturer's instructions. The absorbance of the extracted RNA samples was measured at 280 nm and 260 nm using a Nanodrop (Thermo Scientific NanoDrop 1000 spectrophotometer) to determine their quantity and purity. The extracted RNA samples were converted to complementary DNA (cDNA) using cDNA synthesis kits (Yekta-Tajhiz, Iran) according to the manufacturer's instructions. A real-time PCR system (BioMolecular Systems, Australia) was utilized to assess the CCR2 and DPP9 gene expression using SYBR green master mix (Yekta-Tajhiz, Iran). Real time-PCR amplifications were set up in triplicate with a 10 μl volume containing 5 μl SYBR green master mix, 1 μl of corresponding cDNA and primers ( Table 2). The RT-qPCR was performed at the condition as followed: 94 • C for 15 min, 40 sequential cycles at 95 • C for 10 s and 58 • C for 1 min and the final extension at 72 • C for 10 min. The β-actin gene was also used as an internal control and the levels of the CCR2 and DPP9 expression were calculated by 2 -ΔΔCt formula.

Statistical analysis
All statistical analysis was carried out using SPSS software version 21 (Chicago, IL). Data were analyzed by one-way ANOVA test following Tukey post hoc test. The P values <0.05 were considerd significant.

CCR-2 gene expression alteration in COVID-19 patients
The fold change expression of CCR2 mRNA in the peripheral blood samples from all COVID-19 patients was sugnificantly higher than that in the healthy control group (p < 0.0001) (Fig. 1).
The fold change expression of CCR2 mRNA in the peripheral blood samples from male and female patients with COVID-19 according to their disease severity was indicated in the Fig. 2. In male and female patients with COVID-19, the levels of CCR2 expression significantly differed between moderate, severe, and critical patterns (p < 0.0001) as well as between whole patients and control group (p < 0.0001). Female patients with severe, critical, and moderate forms of COVID-19 expressed higher levels of CCR2 mRNA in comparison with the control group (p < 0.0001) ( Fig. 2A). Similarly, male patients with severe, critical, and moderate forms of COVID-19 expressed higher levels of CCR2 mRNA than that in the control group (p < 0.0001) (Fig. 2B).
The fold change expression of CCR2 mRNA in the peripheral blood samples from patients with different patterns of COVID-19 according to their gender was indicated in the Fig. 3. The female with moderate and critical forms of COVID-19 expressed greater levels of CCR2 than nale patients with same disease forms (p < 0.0001). However, the male patients with severe COVID-19 expressed greater levels of CCR2 than female with same disease form (p < 0.0001) (Fig. 3).

DPP9 gene expression alteration in COVID-19 patients:
The fold change expression of DPP9 mRNA in the peripheral blood samples from all COVID-19 patients was remarkbaly higher compared to healthy control group (p < 0.0001) (Fig. 4).
The fold change expression of DPP9 mRNA in the peripheral blood samples from male and female patients with COVID-19 according to their disease severity was indicated in the Fig. 5. In male and female patients with COVID-19, the expression levels of DPP9 significantly differed between moderate, severe, and critical patterns (p < 0.0001) as well as between whole patients and control group (p < 0.0001). Female patients with severe, critical, and moderate forms of COVID-19 expressed higher levels of DPP9 mRNA in comparison with the control group (p < 0.0001) (Fig. 5A). Similarly, male patients with severe, critical, and moderate forms of COVID-19 expressed higher levels of DPP9 mRNA than that in the control group (p < 0.0001) (Fig. 5B). male and female patients with critical form of COVID-19 expressed significantly higher levels of DPP-9 compared to male and female with severe and moderate COVID-19, respectively (p < 0.0001).
In both male and female groups, an ascending expression of DPP9 mRNA was observed from moderate to severe and critical patterns, indicating a positive association between the expression levels of DPP9 mRNA and disease severity.
The fold change expression of DPP9 mRNA in the peripheral blood samples from patients with different patterns of COVID-19 according to their gender was indicated in the Fig. 6. The female patients with moderate and critical forms of COVID-19 expressed greater levels of DPP9 mRNA than male patients with same disease forms (p < 0.0001). However, the male patients with severe COVID-19 expressed greater levels of CCR2 mRNA than female with same disease form (p < 0.0001) (Fig. 6).

Discussion
Aberrant production of chemokines and impaired activation of inflammasomes play a fundamental role in the pathogenesis of COVID-19. Here, the increased mRNA expression of CCR2 and DPP9 was observed in the peripheral blood samples from all COVID-19 patients compared to the healthy control group. CCR2 acts as a receptor for several chemokines, especially MCP-1. Previous studies have demonstrated that COVID-19 is associated with dynamic changes in CCR2 and DPP9 gene expression (Schmiedel et al., 2020;Pairo-Castineira et al., 2021;Singh et al., 2021). MCP-1/CCR2 axis can participate induction of anti-virus immune responses and promotion of deleterious hyperinflammatory responses during COVID-19. Epithelial cell derived-MCP-1 can recruit CCR2 expressing cells (such as NK cells, monocytes, macrophages and DCs) into the SARS-CoV-2-infected respiratory system . The recruited immune cells can limit the viral infection through several mechanisms, for instance interferon production (Vanderheiden et al., 2021). In a mouse model of SARS-CoV-2 infection, it was found that early CCR2 signaling restricts the viral burden in the lung. CCR2 signaling promotes the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Mice lacking CCR2 showed higher viral loads in the lungs and increased lung viral dissemination .
Elevated levels of MCP-1 (a CCR2 ligand) was reported in COVID-19 patients, especially in the critical form, promoting inflammatory responses through binding to CCR2 . Indeed, MCP-1 is a chemokine among cytokine storm-related chemokines contributing to the tissue inflammation and even organ failure (Jafarzadeh et al., 2020). Thus, targeting the MCP-1/CCR2 axis can attenuate the unwanted inflammatory responses. The selective inhibition of the MCP-1/CCR2 pathway using various antagonist agents has been reported in different clinical trials with inconsistent results (Bianconi et al., 2018). Severe COVID-19 patients displayed greater expression of CCR2 compared to moderate patients, while the expression levels of Table 2 Used primers for RT-qPCR.

Gene name
Sense 5 ′ to 3 ′ Anti-sense 5 ′ to 3 ′ DPP-9 5 ′ -CGAGGTGGAGGTCATTCA- Fig. 1. Comparison of the expression of CCR2 in the peripheral blood samples between total patients and healthy control group. * Significant difference in different groups (p < 0.0001). Data are presented as fold change.
CCR2 was reduced in the critical patients compared to severe COVID-19 patients. The reason for these observations remains to be explained in future studies. Perhaps, the greater tissue leukocytes infiltration in critical COVID-19 could be an account for reduced expression of CCR2 in the peripheral blood. DPP9 acts as an inhibitor of inflammasome (Okondo et al., 2017;Okondo et al., 2018;Zhong et al., 2018). In agreement with our findings, Wang et al. also reported the elevated DPP9 expression in peripheral blood from COVID-19 patients compared to healthy individual and bacterial-infected patients. They have reported that the DPP9 expression increased from day 0 compared to 7 days and 14 days (Brandi, 2021). After that the severity of COVID-19-related symptoms were dramatically improved (Brandi, 2021). Here, we observed an ascending expression of DPP9 mRNA from moderate to severe and critical patterns. It can be speculated that elevated expression of the DPP9 in more serious forms of COVID-19 may be a homeostatic response to modulate deleterious inflammatory responses.
The results of the present study indicate that female patients with moderate and critical forms of COVID-19 expressed greater levels of CCR2 and DPP9 mRNA than male with same disease forms. However, the male patients with severe COVID-19 expressed greater levels of CCR2 and DPP9 mRNA than female with same disease form. It has been reported that COVID-19-related mortality and morbidity are greater in male patients compared to female (Rehman et al., 2021;Chen et al., 2020). Moreover, profound differences have been indicated between male and female concerning the immunologic and inflammatory responses (Rehman et al., 2021). Whether these differences can influence chemokine responses (such as MCP-1/CCR2 pathway) and regulatory mechanisms (such as DPP9) need to be described in future investigations.
Our study may have several limitations: Firstly, in order to elucidate the role of CCR2/MCP-1 pathway, it was necessary to measure the levels of MCP-1 in sera/plasma samples from patients. Previously, MCP-1 has   3. Comparison of CCR-2 gene expression between female and men patients with various forms of COVID-19. Significant difference in different groups (**p < 0.0001 and *p < 0.05). Data are presented as fold change. Fig. 4. Comparison of the expression of DDP9 in the peripheral blood samples between total patients and healthy control group. * Indicate Significant difference in different groups (p < 0.0001). Data are presented as fold change.
been introduced as a biomarker of COVID-19 severity, so that the highest levels of MCP-1 were observed in the patients with critically form promoting inflammatory responses via binding to CCR2 Cabaro et al., 2021). Secondly, in order to elucidate the role of DPP9-related pathway, it was necessary to measure the expression levels of DPP9-related molecules in samples from patients. As mentioned NLRP3 inflammasome can acts an activator of the DPP9, which in turn can inhibit the activity of inflammasome (Okondo et al., 2017;Okondo et al., 2018;Zhong et al., 2018). It has been indicated that SARS-CoV-2 activates NLRP3 inflammasome through induction of P2RX7, releasing of various types of DAMPs from dead cells, and raising in angiotensin II levels . However, inability of DPP9 to control inflammasome may partly contribute to progression of inflammatory responses. Thirdly, evaluation of the influence of the gene polymorphisms on the expression of CCR2 and DPP9 was not a part of our study. As the polymorphisms within the CCR2 and DPP9 can influence their expression, the association of CCR2 and DPP9 gene polymorphisms with the expression levels of these factors and with COVID-19 severity remains to be elucidated in future studies.

Conclusion
In summary, our study shows that the expression of DPP9 and CCR2 was substantially increased in COVID-19 patients with moderate, severe, and critical forms of disease. The expression of both CCR2 and DDP9 was also influenced by the COVID-19 pattern, so that the greatest expression levels of DPP9 and CCR2 was observed in severe forms of disease. Considerable differences were also observed between male and female patients with different disease pattern, concerning the DPP9 and CCR2. Therefore, we further suggest to consider the gender of patients and disease severity in management of COVID-19.