388 J Contemp Med Sci | Vol. 8, No. 6, November-December 2022: 388–394 Original Circulating Levels of Interferon Regulatory Factor-5 Correlates with Disease Activity in Systemic Lupus Erythematosus Iraqi Patients Zainab M. Dahham*, Namir I. A. Haddad Department of Chemistry, College of Science, University of Baghdad, Baghdad, Iraq. *Correspondence to: Zainab M. Dahham (Email: zainab@sc.uobaghdad.edu.iq) (Submitted: 11 August 2022 – Revised version received: 10 September 2022 – Accepted: 17 October 2022 – Published online: 26 December 2022) Abstract Objectives: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by a diversity of the phenotypes among the patients. SLE is still one of the great challenges due to the lacking of specific biomarkers for diagnosis, assessing disease activity, and prediction of response to therapy. This study aimed to investigate the role of circulating levels of IRF5 protein in sample of SLE Iraqi patients and its correlation with disease activity, to identify a potential immunological biomarker to mirror disease activity. Methods: Blood samples were taken from 59 participants diagnosed with SLE cases classified according to the American College of Rheumatology (ACR) criteria. They were scored through the SLE disease activity index 2000 (SLEDAI-2K) to estimate the disease activity, and according to it they were subdivided into “SLE-1 group” (SLEDAI-2k ≤5), and “SLE-2 group” (SLEDAI-2k >5), as well as age and gender matched healthy control group. Circulating levels of IRF5 protein were measured in sera samples by ELISA method. Results: Our result revealed that the circulating levels of IRF5 protein were significantly higher in the SLE-2 group rather than control group (P < 0.01), while there was a non-significant difference between SLE-1 group and control group (P > 0.05), as well as between both SLE patient groups. Moreover, the circulating IRF5 protein levels were found to be correlated positively and significantly with disease activity index in both SLE patient groups. The correlation between the circulating levels of IRF5 protein with other parameters revealed that a significant positive correlation was found in SLE-1 group with ESR and globulins, and negative correlation with Hb and (albumin/globulin) ratio, while in SLE-2 group were positively correlated with urea, creatinine, and uric acid. The analysis of receiver operator curves (ROC) for circulating levels of IRF5 protein in SLE-1 and SLE-2 groups showed a good accuracy to distinguish SLE patients from healthy individuals (AUC = 0.758, sensitivity = 65.5%, and specificity = 69%,), and (AUC = 0.788, sensitivity = 77.3%, and specificity = 72.0%,), respectively. Conclusion: The circulating levels of IRF5 protein correlate with disease activity in SLE patients reflects the possibility of using it as a potential immunological biomarker for diagnosis, and monitoring the disease activity. Keywords: IRF5 protein, lupus erythematosus, systemic, Iraqi patients ISSN 2413-0516 Introduction Systemic lupus erythematosus (SLE) is a chronic multi-system autoimmune disease. It is characterized by the production of excessive auto-antibodies due to break of immune system tol- erance to self-antigen.1-3 This subsequently leads to formation of circulating immune complexes, and immunologically mediated tissues injury.4-6 SLE is a heterogenous disease with a wide range of clinical manifestations and immunological dis- orders. Although the etiology and pathogenesis of SLE are still obscure, several lines of evidence documented that immune disorders may be caused by genetic susceptibility and/or envi- ronmental factors.7-9 The incidence of SLE primarily afflicts women in the reproductive years rather than men, with female to male ratio of approximately 9:1.10,11 The diversity in the clin- ical manifestations among the patients is a great obstacle and might reflect the differences in underlying pathogenesis. SLE is still one of the great challenging for physicians and investi- gators. For more accurate diagnosis it is necessary to find a new reliable and specific biomarker for SLE.12-14 Interferon reg- ulatory factor-5 (IRF5) is a member of the IRF family of the transcription factors. It encodes a 60-63-KDa polypeptides, acts as a regulator of the production of numerous pro-inflam- matory cytokines, including type I interferon IFNs (alpha and beta), IL-12, IL-6, IL-23, and tumor necrosis factor alpha (TNF-α).15,16 It has been shown that the immune response against viral, fungal, and bacterial infections is regulated by IRF5.17 Several recent studies revealed important roles of IRF5 in innate and adaptive immunity, cell growth regulation, apop- tosis, and macrophage polarization.18 Many genome-wide association studies document that there is a robust correlation between IRF5 SNPs and SLE, and that IRF5 high-risk variants play an important role in SLE pathogenesis.19 Numerous studies reported that IRF5 risk variants generally correlated with elevated IRF5 expression levels in SLE blood cells, and with interferon-alpha activity in SLE patients.20 To date, there are few studies about endogenous and extracellular IRF5 pro- tein. Wang and his colleagues (2018), they determined the level of IRF5 protein in WBCs of community-acquired pneu- monia (CAP) patients and healthy donors using flowcytom- etry techniques.21 Idborg et al. (2019), who confirm the presence of extracellular IRF5 protein in circulation. They measured the concentration of it in plasma of SLE patients and age matched healthy controls. The techniques that used are immunoprecipitation followed by Mass spectrometry (IP-MS) and ELISA method.22 This study aimed to determine the con- centration of circulating IRF5 protein in sera of sample of Iraqi SLE patients, and examine its correlation with disease activity to identify its predictive value using ROC analysis for SLE diagnosis as a potential immunological biomarker reflects dis- ease severity. Materials and Methods Subjects A total of 59 patients diagnosed with SLE disease (56 females and 3 males) were recruited from the rheumatology unit of Baghdad Teaching Hospital between November 2020 and Feb- ruary 2021. The SLE patients had met the 1997 American mailto:zainab@sc.uobaghdad.edu.iq 389J Contemp Med Sci | Vol. 8, No. 6, November-December 2022: 388–394 Z.M. Dahham et al. Original Circulating Levels of Interferon Regulatory Factor-5 Correlates with Disease Activity in Systemic Lupus College Rheumatology (ACR) revised criteria for the classifi- cation of SLE.23 Patients with other autoimmune disease, malignant disease, kidney disease, liver disease, alcohol intake, cigarette smoking, and other acute infections were excluded. Full history was taking, clinical examination and all the required routine laboratory tests were performed for all patients in order to assessment the disease activity score using SLE disease activity index 2000 (SLEDAI-2K) score,24 and according to it they were subdivided into “SLE-1 group” (SLE- DAI-2k ≤5) and “SLE-2 group” (SLEDAI-2k >5). A total of 29 age matched healthy controls were also recruited with no evi- dence of any chronic medical illness. Samples Collections Venous blood specimens of 5 ml were drawn after overnight fasting from each participant, then the blood was immediately divided into two portions. The first one (2 ml) was transferred into tube containing K3EDTA, and they were stirred gently for a few seconds to avoid blood clotting and they were used for hematological parameters determination. The reminder blood was transferred into a glass tube with a gel separator. The blood samples were allowed to clot for 10 minutes at 37oC in a water bath, then they were centrifuged at 3000 × g for 10 min- utes. The obtained clear serum was dispensed in several ali- quots, and stored frozen at –20oC until being used to estimate the different parameters included in the study. Hemolyzed sera were excluded. Determination of Circulating Levels of IRF5 Protein IRF5 protein levels were estimated in sera samples using Human IFR5 (interferon regulatory factor5) ELISA kit was supplied by (My BioSource Company, USA) according to manufacturer’s protocol. Laboratory Testing Complete blood count (CBC) was done by Abbott Hema- tology auto-analyzer (Cell-DYN-Ruby, USA). Erythrocyte sedimentation rate (ESR) was determined by the Westergren method. General urine examination (GUE) was determined by routine techniques. Protein urea was quantified by 24-h urine collections. Anti-nuclear antibodies (ANA) and ds-DNA antibodies (ds-DNA) were determined using ELISA method by Naissa Immuno auto-analyzer (Neomedica, Europe). While C3 and C4 concentrations were determined using turbidim- etry method by Hipro Immuno auto-analyzer (Hipro, China). Other biochemical tests were performed by colorimetric methods according to the manufacturer’s instructions using commercial kits. Statistical Analysis The results were presented as mean ± standard deviation (mean ± SD). The differences among the studied groups were calculated by applying analysis of variance one-way (ANOVA), and followed by post hoc Tukey analysis to test the differences between every two groups within ANOVA. The degree of cor- relation between parameters was calculated by Pearson’s cor- relation test. The percentage of significance was obtained by r and P values. The P-value is considered significant if it is < 0.05, and highly significant if it ≤0.01. Receiver Operator Characteristics curve (ROC) analysis was constructed for cir- culating levels of IRF5 protein to estimate its diagnostic yield for SLE disease, the area under the curve were considered exceptional (1–0.9), excellent (0.9–0.81), good (0.8–0.71), fair (0.7–0.61), and poor (0.6–0.5). The statistical analyses were performed using software statistical package for social science (SPSS) version 26.0 (IM SPSS, Chicago, IL, USA) and GraphPad Prism, version. 9.3.1 (San Diego, California, USA). Results The demographic data, clinical and Immunological character- istics of the 88 participants, 59 patients diagnosed as SLE according to the American College Rheumatology (ACR) cri- teria, and 29 healthy controls are described in Table 1. The treatment with medication that used at time of enrollment were also mentioned. It is clear from the results in Table 1 that among the 59 SLE patients, 95% (M:F = 3:56) are females. As for organ involvement, 45 patients (76.2%) had immunological disor- ders, and 31 patients (52.5%) had hematological disorders. The next common manifestations were arthritis in 25 patients (42.3%) and oral ulcers in 22 patients (37%), followed by renal disorders in 16 patients (27.1%). As for medications, most of the SLE patients under therapy, 48 patients (81.3%) with Pred- nisolone treatment, 43 patients (72.8%) with Hydroxychloro- quine treatment, and 15 patients (25.4%) with Azathioprine treatment. There were no statistical differences in age, gender, and BMI among the three studied groups (SLE-1, SLE-2, and control), as well as in disease duration between the SLE patient groups (P > 0.05). However, there is a significant difference in SLEDAI–2K score between the SLE patient groups (P < 0.05). The number of SLE patients with family history in SLE-1 group and SLE-2 group are 4 (14%) and 8 (27%), respectively. On the other hand, all controls with no family history. To investigate the role of circulating levels of IRF5 protein in SLE pathogenesis, sera samples of SLE patients and controls were analyzed. The results showed that circulating levels of IRF5 protein were significantly increased in SLE-2 group with a mean of 1.75 ± 0.65 ng/ml as compared with control group with a mean of 1.24 ± 0.19 ng/ml (P < 0.01). Upon comparison between SLE-1 group and control group the results show a slight increase of circulating levels of IRF5 protein in SLE-1 group with a mean of 1.48 ± 0.65 ng/ml than control group. But this increase was statistically non-significant, as well as non-significant difference was observed between SLE patient groups as shown in Figure 1. Moreover, the circulating levels of IRF5 protein in SLE-1 and SLE-2 groups were significantly and positively correlated with disease activity index (SLEDAI-2K), (r = 0.441, P = 0.017) and (r = 0.502, P = 0.005), respectively as shown in (Figure 2A and 2B). Additionally, we found a significant positive correlation between circulating levels of IRF5 protein and relative expres- sion of IRF5 mRNA levels in both SLE-1 and SLE-2 groups, (r = 0.887, P < 0.0001) and (r = 0.847, P < 0.0001), respectively. Data not shown as it is another part of our project and it is under publication elsewhere.25 The correlation of circulating levels of IRF5 protein with other parameters in both SLE patient groups were summarized in Table 2. Circulating levels of IRF5 protein in SLE-1 group were positively and signifi- cantly correlated with ESR (r = 0.452, P = 0.014) and globulins (r = 0.463, P = 0.011), and negative correlation with Hb (r = –0.459, P = 0.012) and (albumin/globulin) ratio (r = –0.484, 390 J Contemp Med Sci | Vol. 8, No. 6, November-December 2022: 388–394 Circulating Levels of Interferon Regulatory Factor-5 Correlates with Disease Activity in Systemic Lupus Original Z.M. Dahham et al. Table 1. Baseline characteristics of SLE patients and healthy controls Characteristic Total SLE patients SLE-1 group SLE-2 group Control group P-value Demographic data Samples number 59 29 30 29 - Age (year), mean ± SD (range) 34.59 ± 10.96 (14–55) 34.53 ± 10.8 (18–55) 34.66 ± 11.2 (14–53) 33.03 ± 9.6 (18–52) 0.81 BMI (Kg/m2), mean ± SD 27.17 ± 6.1 26.06 ± 6.3 28.32 ± 5.5 26.35 ± 5.6 0.285 Gender Female, n (%) Male, n (%) 56 (95%) 3 (5%) 27 (93%) 2 (7%) 29 (97%) 1 (3%) 26 (90%) 3 (10%) 0.574 Disease duration (year), mean (range) 4.85 (0.1–33) 4.64 (0.3–23) 5.04 (0.1–33) - 0.956 Family history with SLE 12 (20%) 47 (80%) 4 (14%) 25 (86%) 8 (27%) 22 (73%) - -Yes, n (%) No, n (%) Clinical and immunological manifestations Vasculitis, n (%) 2 (3.3%) 0 (0%) 2 (6.6%) - - Arthritis, n (%) 25 (42.3%) 10 (34.5%) 15 (50%) - - Myositis, n (%) 0 (0%) 0 (0%) 0 (0%) - - Pleurisy, n (%) 3 (5%) 0 (0%) 3 (10%) - - Proteinuria, n (%) 12 (20.3%) 0 (0%) 12 (40%) - - Hematuria, n (%) 2 (3.3%) 0 (0%) 2 (6.6%) - - Urinary casts, n (%) 2 (3.3%) 0 (0%) 2 (6.6%) - - Oral ulcers n (%) 22 (37.2%) 10 (0%) 12 (43.3%) - - Alopecia, n (%) 11 (18.6%) 4 (13.8%) 7 (33.3%) - - Fever, n (%) 10 (16.9%) 3 (10.3%) 7 (33.3%) - - New rash, n (%) 11 (18.6%) 5 (17.2%) 6 (36.6%) - - Thrombocytopenia, n (%) 12 (20.3%) 3 (10.3%) 9 (30%) - - Leucopenia, n (%) 4 (6.7%) 3 (10.3%) 1 (3.3%) - - Anemia, n (%) 29 (49.1%) 11 (37.9%) 18 (60%) - Low complement n (%) 18 (30.5%) 3 (10.3%) 15 (50%) - - (+) ANA, n (%) 40 (67.7%) 15 25 - (+) ds-DNA antibodies, n (%) 36 (61%) 15 (51.7%) 21 (70%) - - SLEDAI-2K, mean ± SD (range) 7.37 ± 3.94 (2–18) 4.1 ± 0.72 (2–5) 10.53 ± 3.08 (6–18) - <0.01 Medications Prednisolone Yes, n (%) 48 (81%) 11 (19%) 22 (76%) 7 (24%) 26 (87%) 4 (13%) - - Treatment No, n (%) Hydroxychloroquine Yes, n (%) 43 (73%) 16 (27%) 19 (66%) 19 (34%) 24 (80%) 6 (20%) - - Treatment No, n (%) Azathioprine Yes, n (%) 15 (25%) 44 (75%) 8 (28%) 21 (72%) 7 (23%) 23 (77%) - - Treatment No, n (%) Methotrexate Yes, n (%) 3 (5%) 56 (95%) 1 (3%) 28 (97%) 2 (7%) 28 (93%) - - Treatment No, n (%) The collected data was analyzed by mean ± SD (mean ± standard deviation), range (minimum-maximum), or number (percentage). P-value was used for the comparison among the three studied groups (SLE-1, SLE-2, and control). P > 0.05 = non-significant differences, P < 0.05 = significant differences, P ≤ 0.01 = high significant differences. BMI, body mass index; ANA, anti-nuclear antibodies; ds-DNA antibodies, double strand deoxy nucleic acid antibodies; SLEDAI-2K, Systemic lupus erythematosus disease activity index 2000. 391J Contemp Med Sci | Vol. 8, No. 6, November-December 2022: 388–394 Z.M. Dahham et al. Original Circulating Levels of Interferon Regulatory Factor-5 Correlates with Disease Activity in Systemic Lupus P = 0.008). While circulating levels of IRF5 protein in SLE-2 group were positively correlated with urea (r = 0.632, P < 0.0001), creatinine (r = 0.751, P < 0.0001), and uric acid (r = 0.595, P = 0.001). In order to estimate the ability of circulating levels of IRF5 protein to distinguish the active SLE patient from healthy subjects as a diagnostic biomarker. We analyzed it using ROC curve analysis. The result showed that the circu- lating levels of IRF5 protein in SLE-1 and SLE-2 groups had a good ability to discriminate SLE patients from healthy per- sons. By which the AUC, sensitivity and specificity were (0.758, 65.5%, 69.0%), and (0.778, 72.4%, 70.0%) respectively, at the cut off value of 1.35 (ng/ml), and 1.365 (ng/ml) respec- tively, which was the good value of SLE correct prediction, as shown in (Fig 3A and 3B). Discussion This study has been prepared to evaluate the circulating levels of IRF5 protein in a sample of SLE Iraqi patients, and to examine the association with disease activity index and other parameters. Moreover, applying the ROC curve analysis in Fig. 1 Circulating levels of IRF5 protein among the studied groups. The results were expressed as mean ± SD (mean ± standard deviation), P > 0.05 = statistically non-significant differences, * Statistically significant differences at P < 0.05, ** Statistically significant differences at P ≤ 0.01. Fig. 2 (A and B), the correlation between Circulating levels of IRF5 protein (ng/ml) and SLEDAI-2K score in SLE-1 group and SLE-2 group, respectively. r, Pearson coefficient. P > 0.05 = statistically non-significant correlation, *Statistically significant correlation at P < 0.05, ** Statistically significant correlation at P ≤ 0.01. Table 2. The correlation between circulating levels of IRF5 protein (ng/ml) with other biochemical parameters Parameter Circulating levels of IRF5 protein (ng/ml) SLE-1 group n = 29 SLE-2 group n = 30 r P-value r P-value Demographic data Age (year) 0.353 0.06 0.124 0.515 Gender (F/M) –0.259 0.175 –0.76 0.689 BMI (Kg/m2) 0.218 0.255 0.084 0.659 Disease duration (year) 0.098 0.612 -0.001 0.997 Family history with SLE –0.222 0.309 0.409 0.058 Hematological parameters WBC X 103 /UL –0.297 0.118 0.353 0.060 RBC X 106 /UL –0.245 0.201 –0.212 0.261 Hb (gm/dl) –0.459 0.012* –0.269 0.151 PLT X 103 /UL 0.153 0.429 0.366 0.051 ESR (mm/1 hr) 0.452 0.014* 0.106 0.546 Biochemical parameters Urea (mg/dl) 0.145 0.452 0.632 <0.0001** Creatinine (mg/dl) 0.04 0.838 0.751 <0.0001** Uric acid (mg/dl) 0.353 0.060 0.595 <0.001** GOT (U/L) -0.069 0.723 0.352 0.057 GPT (U/L) 0.224 0.242 0.184 0.0331 ALP (U/L) 0.366 0.051 -0.51 0.79 Total serum protein (g/l) 0.207 0.281 -0.172 0.364 Serum albumin (g/l) -0.394 0.063 -0.180 0.340 Globulins (g/l) 0.463 0.011* 0.011 0.954 Albumin /globulins -0.484 0.008** -0.098 0.608 Total cholesterol (mg/dl) -0.052 0.787 0.328 0.077 Tri glyceride (mg/dl) 0.065 0.737 0.272 0.145 VLDL (mg/dl) 0.063 0.747 0.272 0.145 HDL (mg/dl) -0.214 0.264 0.098 0.607 LDL (mg/dl) -0.029 0.879 0.302 0.105 r, Pearson coefficient. P > 0.05 = statistically non-significant correlation, *Statistically significant correlation at P < 0.05, **Statistically significant correlation at P ≤ 0.01. BMI, body mass index; WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; PLT, platelets; ESR, erythrocyte sedimentation rate; GOT, glutamate oxaloacetate transaminase; GPT, Glutamate Pyruvate Transaminase; ALP, alkaline phosphatase; vLDL, very low-density lipopro tein; HDL, high-density lipoprotein; LDL, low-density lipoprotein. 392 J Contemp Med Sci | Vol. 8, No. 6, November-December 2022: 388–394 Circulating Levels of Interferon Regulatory Factor-5 Correlates with Disease Activity in Systemic Lupus Original Z.M. Dahham et al. order to predict the ability of it for diagnosis of SLE patients as an immunological biomarker. Our result revealed that circu- lating levels of IRF5 protein were found to be increased in both SLE-1 and SLE-2 groups as compared with healthy controls, and correlated positively and significantly with disease activity index. IRF5 is a transcription factor plays an important role in inflammatory response. it is likely a key regulator of the toll like receptors (TLRs). In the case of the unstimulated cell, IRF5 is generally localized in the cytoplasm as a monomer. Activation of the above receptors leads to cascading signals. IRF5 undergoes posttranslational modification, which eventu- ally leads to homodimerization, a critical event prior to nuclear translocation.17 The extracellular protein of IRF5 still has an unknown function in the circulation and away from complete transcription factors and other nuclear molecules. It can have an unknown function or may regardless of the function.22 The functions of extracellular and intracellular protein might be different and unusual secretion is also probable.26 Our finding is in agreement with Idbord et al., who found that circulating levels of IRF5 protein were significantly higher in SLE patients compared with control individuals. They reported that the high level of IRF5 protein in plasma samples of SLE patients may be reflected the increase of cell death during apoptosis clearance in SLE patients.22 In other hands, we cannot only be clarified by this reason because the reports of transcription factor in circulation are limited.27,28 There is no information about the function of extracellular of IRF5 protein. However, the fact that IRF5 may be present in microparticles, known to mediate cell-cell signaling thus further studies are needed about circulating IRF5 protein.22 The expression of IRF5 gene is significantly raised in peripheral mononuclear cells (PBMC) from SLE patients as compared to age-matched healthy indi- viduals, and this can stimulate the expression of type I inter- feron.29 The elevated serum levels of IFN- alpha have been shown to be associated with the activity and severity of SLE disease. These findings support to explain our results about the positive correlation between circulating levels of IRF5 protein and disease activity.30 Numerous studies in different countries population have showed that the hematological abnormalities are present in most of SLE patients. The common hematolog- ical syndrome in SLE patients is anemia, which is most often owing to the anemia of chronic disease. ESR levels were signif- icantly increase in SLE patients during SLE flare and infec- tions as compared to healthy controls.31-33 Many studies indicated that ESR considered an important factor in assessing SLE disease activity, as ESR elevates when disease activity increase. It is believed that the reason for the high rate of erythrocyte sedimentation is the decrease in the concentration of proteins in the blood plasma, as well as the change in the shape of the surface of the erythrocytes and their adhesion to each other.34 These findings may be explaining our results about the negative correlation between Hb and circulating IRF5 protein, as well as the positive correlation between it and ESR level. SLE is characterized by raised levels of autoreactive antibodies and gamma globulin. The production of autoanti- bodies requires the synthesis of gamma globulin and this led to increase the globulin levels in blood SLE patients.35,36 Our results about positive proteinuria in SLE patients as shown in Table 1 are in agreement with previous studies that documented the presence of protein in urine in SLE patients.37,38 Serum albumin was determined in SLE patients as a part of routine biochemical tests. The decrease of serum albumin levels in SLE patients may be caused by elevating albumin catabolism as a result of chronic inflammation and/or because of poor diet from proteins content and calories. More- over, the common manifestation in SLE patients is nephritis which characterized by attack the kidney membranes due to the presence of auto-antibodies against these membranes. Consequently, membranes are disrupted and impaired its Group AUC SE P-value 95% IC Cut-off value Sensitivity Specificity PPV NPV Accuracy SLE-1 0.758 0.064 0.001 0.633-0.883 1.350 65.5% 69.0% 67.9% 66.7% 67% SLE-2 0.778 0.06 <0.001 0.659-0.896 1.365 72.4% 70.0% 72.4% 70.0% 71.1% Fig. 3 (A&B) Receiver operator curve (ROC) analysis for the predictive value of circulating levels of IRF5 protein in SLE-1 (n = 29) and SLE-2 (n = 30) versus healthy controls (n = 29), respectively. AUC = area under the curve, SE = standard error, CI = confidence interval, PPV = positive predictive value, NPV = negative predictive value. 393J Contemp Med Sci | Vol. 8, No. 6, November-December 2022: 388–394 Z.M. Dahham et al. Original Circulating Levels of Interferon Regulatory Factor-5 Correlates with Disease Activity in Systemic Lupus filtration ability. In normal condition the filtering membranes do not allow albumin and another blood proteins to be missing in the urine. However, in lupus nephritis the protein loss in urine which in turn lowers serum albumin concentrations,39,40 and this lead to decrease the ratio of albumin/globulin. Kwon et al. (2018) found that the (albumin/globulin) ratio in SLE with nephritis was lower than in SLE without nephritis.36 All above findings supported our results about the positive corre- lation between circulating level of IRF5 protein and globulins, as well as a negative correlation with the ratio of (albumin/ globulin). Our results of the positive correlation with urea, creatinine, and uric acid could be explained by the existence of some patients with lupus nephritis as shown in Table 1. Con- sequently, the existence of any criterion correlated with kidney disorder for example proteinuria and hematuria lead to raise the score of disease activity. Therefore, patients with lupus nephritis graded the high score of disease activity among the patients, subsequently increasing their levels of circulating IRF5 protein. ROC curve analysis, it showed that circulating levels of IRF5 protein in SLE-1 and SLE-2 groups could repre- sent a good predictor for SLE diagnosis. Conclusion Our study results suggests that the IRF5 may play an impor- tant role in SLE pathogenesis, and IRF5 may be useful in diag- nosis of SLE. The circulating levels of IRF5 protein associated with disease flare in SLE patients reflect the possibility of using it as a potential biomarkers for diagnosis, monitoring the dis- ease course and response to therapy. Moreover, the ELISA method is more rapid and inexpensive rather than real time PCR for determination of IRF5 gene expression. Conflict of Interest None.  References 1. La Paglia, G.M.C., Leone, M.C., Lepri, G., Vagelli, R., Valentini, E., Alunno, A. and Tani, C., 2017. One year in review 2017: systemic lupus erythematosus. Clin Exp Rheumatol, 35(4), pp. 551–561. 2. Idborg, H. and Oke, V., 2021. Cytokines as biomarkers in systemic Lupus Erythematosus: Value for diagnosis and drug therapy. International Journal of Molecular Sciences, 22(21), p.11327. 3. Abdulridha, R.H., Saud, A.M. and Alosami, M.H., 2022. Evaluation of Interferon Alpha (IFN-α) in Women with Systemic Lupus Erythematosus in Iraq. Iraqi Journal of Science, pp. 4225–4233. 4. Hellquist, A., Järvinen, T.M., Koskenmies, S., Zucchelli, M., Orsmark-Pietras, C., Berglind, L., Panelius, J., Hasan, T., Julkunen, H., D’AMATO, M.A.U.R.O. and Saarialho-Kere, U., 2009. Evidence for genetic association and interaction between the TYK2 and IRF5 genes in systemic lupus erythematosus. The Journal of Rheumatology, 36(8), pp. 1631–1638. 5. Trentin, F., Zucchi, D., Signorini, V., Elefante, E., Bortoluzzi, A. and Tani, C., 2021. One year in review 2021: systemic lupus erythematosus. Clin Exp Rheumatol, 39(2), pp. 231–241. 6. Hiba S. Ahmed, Hind S. Ahmed, Ali Ad’hiah, 2020. Interleukin-1 single nucleotide polymorphisms and risk of systemic lupus erythematosus among Iraqi patients. Meta Gene 23 (100640). 7. Quan, W., An, J., Li, G., Qian, G., Jin, M., Feng, C., Li, S., Li, X., Xu, Y. and Hu, X., 2021. Th cytokine profile in childhood-onset systemic lupus erythematosus. BMC Pediatrics, 21(1), pp. 1–10. 8. Katsuyama, T., Tsokos, G.C. and Moulton, V.R., 2018. Aberrant T cell signaling and subsets in systemic lupus erythematosus. Frontiers in immunology, 9, p. 1088. 9. Jasem, M.A., 2007. Hyperprolactenemia in Women with Systemic Lupus Erythematusus. Baghdad Science Journal, 4(4). 10. Urrego, T., Ortiz-Reyes, B., Vanegas-García, A.L., Muñoz, C.H., González, L.A., Vásquez, G. and Gómez-Puerta, J.A., 2020. Transferrina y ceruloplasmina en orina de pacientes con lupus eritematoso sistemico.? Son utiles para diferenciar pacientes con nefritis lupica? Reumatología Clínica, 16(1), pp. 17–23. 11. Tselios, K., Gladman, D.D., Touma, Z., Su, J., Anderson, N. and Urowitz, M.B., 2019. Disease course patterns in systemic lupus erythematosus. Lupus, 28(1), pp. 114–122. 12. Liu, C.C., Kao, A.H., Manzi, S. and Ahearn, J.M., 2013. Biomarkers in systemic lupus erythematosus: challenges and prospects for the future. Therapeutic Advances in Musculoskeletal Disease, 5(4), pp. 210–233. 13. Capecchi, R., Puxeddu, I., Pratesi, F. and Migliorini, P., 2020. New biomarkers in SLE: from bench to bedside. Rheumatology, 59(Supplement_5), pp. v12-v18. 14. Rekvig, O.P., 2018. Systemic lupus erythematosus: definitions, contexts, conflicts, enigmas. Frontiers in Immunology, 9, p. 387. 15. Mancl, M.E., Hu, G., Sangster-Guity, N., Olshalsky, S.L., Hoops, K., Fitzgerald- Bocarsly, P., Pitha, P.M., Pinder, K. and Barnes, B.J., 2005. Two discrete promoters regulate the alternatively spliced human interferon regulatory factor-5 isoforms: multiple isoforms with distinct cell type-specific expression, localization, regulation, and function. Journal of Biological Chemistry, 280(22), pp. 21078–21090. 16. Cevik, O., Li, D., Baljinnyam, E., Manvar, D., Pimenta, E.M., Waris, G., Barnes, B.J. and Kaushik-Basu, N., 2017. Interferon regulatory factor 5 (IRF5) suppresses hepatitis C virus (HCV) replication and HCV-associated hepatocellular carcinoma. Journal of Biological Chemistry, 292(52), pp. 21676–21689. 17. Wang, M., Lim, K.H. and Chow, K.T., 2019. Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization. JoVE (Journal of Visualized Experiments), (152), p. e60393. 18. Banga, J., Srinivasan, D., Sun, C.C., Thompson, C.D., Milletti, F., Huang, K.S., Hamilton, S., Song, S., Hoffman, A.F., Qin, Y.G. and Matta, B., 2020. Inhibition of IRF5 cellular activity with cell-penetrating peptides that target homodimerization. Science Advances, 6(20), p. eaay1057. 19. Ebrahimiyan, H., Bagheri-Hosseinabadi, Z. and Abbasifard, M., 2021. Interferon regulatory factor 5 in Rheumatoid arthritis and systemic lupus erythematosus. Rheumatology Research, 6(1), pp.1–13. 20. Li, D., Matta, B., Song, S., Nelson, V., Diggins, K., Simpfendorfer, K.R., Gregersen, P.K., Linsley, P. and Barnes, B.J., 2020. IRF5 genetic risk variants drive myeloid- specific IRF5 hyperactivation and presymptomatic SLE. JCI Insight, 5(2). 21. Wang, X., Guo, J., Wang, Y., Xiao, Y., Wang, L. and Hua, S., 2018. Expression levels of interferon regulatory factor 5 (IRF5) and related inflammatory cytokines associated with severity, prognosis, and causative pathogen in patients with community-acquired pneumonia. Medical science monitor: international medical journal of experimental and clinical research, 24, p. 3620. 22. Idborg, H., Zandian, A., Ossipova, E., Wigren, E., Preger, C., Mobarrez, F., Checa, A., Sohrabian, A., Pucholt, P., Sandling, J.K. and Fernandes- Cerqueira, C., 2019. Circulating levels of interferon regulatory factor-5 associates with subgroups of systemic lupus erythematosus patients. Frontiers in Immunology, 10, p. 1029. 23. Hochberg, M.C., 1997. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis and rheumatism, 40(9), pp. 1725–1725. 24. Gladman, D.D., Ibanez, D. and Urowitz, M.B., 2002. Systemic lupus erythematosus disease activity index 2000. The Journal of Rheumatology, 29(2), pp. 288–291. 25. Zainab M. Dahham, Namir I. A. Haddad, Correlation Between Gene Expression of Interferon Regulatory Factor-5 and Disease Activity Index in Systemic Lupus Erythematosus Iraqi Patients. Iraqi Journal of Science (under publication). 26. Radisky, D.C., Stallings-Mann, M., Hirai, Y. and Bissell, M.J., 2009. Single proteins might have dual but related functions in intracellular and extracellular microenvironments. Nature Reviews Molecular Cell Biology, 10(3), pp. 228–234. 27. Raghavan, S., Manzanillo, P., Chan, K., Dovey, C. and Cox, J.S., 2008. Secreted transcription factor controls Mycobacterium tuberculosis virulence. Nature, 454(7205), pp. 717–721. 28. Lomnytska, M., Dubrovska, A., Hellman, U., Volodko, N. and Souchelnytskyi, S., 2006. Increased expression of cSHMT, Tbx3 and utrophin in plasma of ovarian and breast cancer patients. International Journal of Cancer, 118(2), pp. 412–421. 394 J Contemp Med Sci | Vol. 8, No. 6, November-December 2022: 388–394 Circulating Levels of Interferon Regulatory Factor-5 Correlates with Disease Activity in Systemic Lupus Original Z.M. Dahham et al. 29. Feng, D., Stone, R.C., Eloranta, M.L., Sangster‐Guity, N., Nordmark, G., Sigurdsson, S., Wang, C., Alm, G., Syvänen, A.C., Rönnblom, L. and Barnes, B.J., Genetic variants and disease‐associated factors contribute to enhanced interferon regulatory factor 5 expression in blood cells of patients with systemic lupus erythematosus,” Arthritis & Rheumatism: Official Journal of the American College of Rheumatology, vol. 62, no. 2 pp. 562–573, 2010. 30. Jefferies, C.A., 2019. Regulating IRFs in IFN driven disease. Frontiers in immunology, 10, p. 325. 31. Sasidharan, P.K., Bindya, M. and Sajeeth Kumar, K.G., 2012. Hematological manifestations of SLE at initial presentation: is it underestimated? International Scholarly Research Notices, 2012. 32. Newman, K., Owlia, M.B., El-Hemaidi, I. and Akhtari, M., 2013. Management of immune cytopenias in patients with systemic lupus erythematosus— old and new. Autoimmunity reviews, 12(7), pp. 784–791. 33. Ismail Zoair, M., Zaki El-Ghannam, M., Gaber, A.E.A. and Mohammed Soliman, A., 2021. Assessment the role of urine osteoprotegerin as a biomarker in lupus nephritis. Al-Azhar Medical Journal, 50(3), pp. 2201–2212. 34. Majeed, W.A., Jasim, H.M., Farhan, A.A., Ali, A.S. and Gorial, F.I., 2017. Detection of Hematological Biomarkers Associated with Lupus Nephritis in a sample of Iraqi Patients. Journal of Biotechnology Research Center, 11(2). This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v8i6.1299 35. Baum, C.G., Chiorazzi, N., Frankel, S. and Shepherd, G.M., 1989. Conversion of systemic lupus erythematosus to common variable hypogammaglobulinemia. The American Journal of Medicine, 87(4), pp. 449–456. 36. Kwon, O.C., Lee, J.S., Ghang, B., Kim, Y.G., Lee, C.K., Yoo, B. and Hong, S., 2018, December. Predicting eventual development of lupus nephritis at the time of diagnosis of systemic lupus erythematosus. In Seminars in arthritis and rheumatism (Vol. 48, No. 3, pp. 462–466). WB Saunders. 37. Nazri, S.K.S., Wong, K.K. and Hamid, W.Z.W., 2018. Pediatric systemic lupus erythematosus: Retrospective analysis of clinico-laboratory parameters and their association with Systemic Lupus Erythematosus Disease Activity Index score. Saudi Medical Journal, 39(6), p. 627. 38. Al-Sarray, Z.A., Al-Rayahi, I.A., Al-Hafidh, A.H. and Dwayyikh, A.T., 2020. Serum Protein Electrophoresis in Iraqi Systemic lupus Erythematous Patient. Al-Nisour Journal for Medical Sciences, 2(1). 39. Yip, J., Aghdassi, E., Su, J., Lou, W., Reich, H., Bargman, J., Scholey, J., Gladman, D.D., Urowitz, M.B. and Fortin, P.R., 2010. Serum albumin as a marker for disease activity in patients with systemic lupus erythematosus. The Journal of rheumatology, 37(8), pp. 1667–1672. 40. Cojocaru, M., Cojocaru, M.I., Silosi, I. and Vrabie, D.C., 2010. Kidney damage in autoimmune diseases. Journal of Medical Biochemistry, 29(2), pp. 61–65.