INTRODUCTION:

Human cytomegalovirus (HCMV) is an enveloped, double stranded DNA herpes viruses causing asymptomatic latent lifelong infections in 60-90% of human population (1,2). Worldwide, HCMV is the most common reason of congenital infection and recurrent spontaneous abortion (RSA) in gestated women (3, 4). In response to the infection, T cells and macrophages, which are actively enrolled into the endometrium in early gestation, may excrete redundant amounts of Th1 cytokines (5). Actually, the implantation and successful gestation are associated with a predominance of Th2 cytokines (6). It is apparent that primary HCMV infection excite a series of potent cell mediated immune responses at first by innate NK cells, then by adaptive T cells (CD4+ and CD8+) and B cell height avidity neutralizing antibodies (7).

Over lytic infection, HCMV can directly alter innate/intrinsic immune responses like the interferon (IFN) responses (8) in addition to both intrinsic and extrinsic apoptosis pathways (9). The virus encodes proteins which act as receptors to host inflammatory cytokines, leading to diminution localized cytokine efficiency (10). HCMV encodes a numeral of viral homologs of cytokines such as UL146 [interleukin (IL)-8 like] as well as UL111a (vIL-10), an immunosuppressive IL-10 homolog (11, 12). Indeed, IL-10 is a potent inhibitor of Th1 cytokines (such as IL-2 and IFN-γ ) and also suppress inflammatory cytokine production from macrophages and monocytes that results in a reduction in surface MHC class II expression and a decrease of antigen presentation to CD4+ T cells (13,14). Height IL-10 levels act as a regulatory trigger of resolution of the acute phase of viral infection (15, 16).

Secretion of IL-10 from macrophages, CD4+CD25+FoxP3+ regulatory cells (Tregs), and other leukocytes followed by subsequent bound to IL-10 receptors on dendritic cells (DCs) and macrophages has been related to diminutive antigen presentation. Increased T-cell anergy(17,18) as well as reduce the Th1 responses development occur by diminishing Th1 related cytokines (IFN-γ and IL-12) and enhancing Th2 responses by mounting of Th2 related cytokines (IL-4, IL-13 & IL-5) (19,20). IL-10 support proliferation of B-cells, development of plasma cells, and has differential responses regarding isotype switching (21,22). Moreover, elevated IL-10 levels has been connected with increased efficiency of programmed cell death 1 (PD-1) protein and cytotoxic T-cell lymphocyte antigen 4 (CTL4-A) co-receptors on T-cell that play as negative regulators of T-cells, whereas also being connected with reduced CD28 co-receptor activity on T-cells, all of which are correlating with reduced function of effector T-cell (17,18,23). Furthermore, IL-10 is effective at diminishing levels of pro-inflammatory cytokines: IL-6, IL-2, IL-12, IL-1, GM-CSF, IFN-γ and TNF-α, in both stimulatory and naive condition regarding to the presence or absence of adequate antigen (17,24). The drooping of these cytokines diminish leukocyte maturation, induction and inflammation (25).Because there is no local study that deals with the full immunological picture, this study was designed to evaluate immune profile of innate and adaptive immunity in Iraqi women with RSA that may provide a new insight into the future of immunotherapies development and CMV management.

MATERIALS AND METHODS:

Samples were collected from 50 women with RSA proved with CMV infection (mean of age of women ages is 29.54 ranged from 23-38 years) at Consulting Clinic of Al-Emamain Al-Khadhemain Teaching hospital during the period from January to May 2013. Control sample were collected from 30 family-unrelated, apparently healthy women ( ages mean is 28 ranged from 17- 44 years). Five ml of venous blood were collected in plan tubs from each subjects. separated serum from blood samples were divided to many aliquots in Eppendorf tubes and stored at -20C until the immune tests are performed.

Enzyme-linked immune-sorbent assay (ELISA) was used to estimate serum levels of GM-CSF, IL-1α, IL-8 (Immunotech, France), IL-4(Bioscience, England), IL-10, IL-12 and IL-6( Biosource, Belgium ). Single radial immunodiffusion (SRID) assay was used to estimate serum levels of C4 and C3 (Binding site, England).

Statistical analysis:

The softwareSPSS program (version 20) was used for statistical analysis. In order to compare means of the parameter levels between cases and control, student test was used. All levels of measured were expressed as (Mean ± Standard error). In all tests, significance was set at 0.05 (P ≤ 0.05).

RESULTS:

The result of present study showed a significant dropping in both of GM-CSF and IL-6 levels, while there was a significant elevation in IL-8 levels compared to control group. On the other hand, each of IL-1α, IL-12, and IL-4 showed non-significant elevation, while IL-10 showed non-significant dropping compared to control group ( table1).

Table (1) immunological marker in studied group.

Immune factor	Mean ± SE (pg/ ml)		P value
Immune factor	Control	CMV	P value
GM-CSF	36.55 ± 3.06	24.46 ± 1.20	0.05
IL-1α	16.00 ±10.37	25.90 ± 4.09	0.21
IL-8	222.50 ± 81.01	1824.60 ±259.19	0.01
IL-12	27.90 ± 9.79	32.50 ± 8.57	0.93
IL-6	165.40 ± 6.02	62.00 ± 30.87	0.04
IL-4	20.30 ± 2.01	27.90 ± 1.40	0.65
IL-10	10.10 ± 2.06	5.71 ± 2.05	0.60

Abbreviations: GM-CSF, granulocyte-macrophage colony stimulating factor; IL-1α, interleukin-1alpha; IL-8, interleukin-8; IL-12, interleukin-12; IL-6, interleukin-6; IL-4, interleukin-4; IL-10, interleukin-10.

Moreover, serum levels of C4 showed significant dropping, while C3 levels showed non-significant dropping compared to control group (table2)

Table (2): IgG, C3 and C4 levels in studied group.

Immune factor	Mean ± SE (mg/ dl)		P value
Immune factor	Control	CMV	P value
C3	1464.3 ±176.84	1221.6 ± 81.61	0.15
C4	25.69 ± 3.90	18.98 ±1.28	0.00

Abbreviations: C3, complement 3; C4, complement 4.

DISCUSSION:

The present study revealed that success of CMV infection associated with modulation of immune response in Iraqi women with RSA. Regarding GM-CSF, there was a significant elevation of GM-CSF levels in our results. Perriconeet al. reported that GM-CSF levels were significantly elevated in healthy women compared with those of RSA (26). In fact, GM-CSF can be produced by many cells. Main cells of GM-CSF sources are B and T cells, monocyte/macrophage, fibroblasts, and endothelial cells (27). GM-CSF production is catalyzed by various factors, including IL-1 and TNF (28). The ability of GM-CSF to polarize macrophages to M1-like inflammatory macrophages, that produce a set of inflammatory cytokines such as IL-6, IL-12, and IL-1 (29) may explain non-significant elevation in both of IL-1 alpha and IL-12 levels also significant elevation of IL-8 levels in current study. In this context, in study on Iraqi womenwith RSA Al-Dahmoshietal. reported that IL-8 levels were significantly elevated compared to healthy women (30). Other study also reported that IL-8 levels can be detected in women with RSA (31). The inflammatory cytokines, like IL-8, may play a vital role in the mechanism of protease-induced neurogenic inflammation leading to labor or abortions by enrolling neutrophils and lymphocytes in the endometrium (32). In contrast, Koumantaiet al. reported that women with RSA showed decreased level of IL-8 compared to healthy women (33). The elevated levels of IL-8 in RSA women may be due to the liberation of IL-8 from the endometrium and from an increased numbering of habitant mast cells which are degranulated in abortions (34). Degranulation of uterine mast cells after stress exposure of gestated mice, probably leading to liberation of IL-8 and TNF-α that could be implicated in abortions (32). Furthermore, IL-8 stimulate function of neutrophils and elastase action in the intrauterine milieu has been engaged in the mechanisms of rupture of fetal membrane and cervical accomplishment (35).

Regarding IL-6, local study conducted by Alshamari et al. reported that IL-6 level showed non-significant differences between RSA and healthy women (36). However, this result does not consent with current study which revealed significant dropping in RSA group.

IL-6 play a substantial role in down-regulation of cytokines produced by Th1 (37). Polymorphisms of IL-6 are associated with paradoxical effect, some studies reported that IL-6 polymorphism may be connected with RSA (38), while others do not exhibit such association (39) or it might be with defensive effect (40).

In fact, decreased levels of IL-6 were reported in women withmissed abortion, while no differences were shown in threatened abortion women as compared to normal gestated and non-gestated controls (37). Lim et al. reported that IL-6 protein and mRNA expression were decreased in RSA women as compared to heathy group (41).This result consents with present study. Astudy conducted in vitro revealed that activation of mononuclear cells collected from RSA women at the time of abortion produces low level of IL-6 (42). Another study revealed dropping of IL-6 levels in RSA women as compared to control (43). Another study reported that levels of IL-6 increased significantly in both of gestated and non-gestated women with RSA compared to healthy fertile women (44).

In a study conducted in Iraqi normal gestated and RSA women in 1st trimester as well as in the healthy fertile non gestated women, there was a significant elevation in TNF- α and IFN- γ levels (Th1 cytokines) as well as in Th1/TH2 of RSA women than normal gestated and non-gestated women. Moreover, the study found significant elevation in IL-10 and IL-4 (Th2 cytokines) in normal gestated women. Furthermore, RSA was associated with increasing of Th1 cytokines levels; however successful pregnancy was associatedwith anelevation of Th2 cytokines during the first trimester of gestation (45). Non-significant differences in both of IL-4 and IL-10 levels as well as in IL-12 levels may be due to resolution of acute immune response followed the abortion. Th2 to Th1 shift in gestation might be due to one or more agents. In fact, the deficiency of several putative immunomodulatory factors such as PIBF, placental factors, TGF-β2 or IL-10 probably is responsible. Also, it is likely that a poise between IL-4 (favoring Th2 immune response) and IL-12 (favoring Th1 immune response) determines the conclusive effect of the Th2-Th1 dichotomy over an immune response (46).

Related to C3 and C4 complements, in a local study on Iraqi women with RSA conducted by Al-Khayatet al. revealed that each of C3 and C4 levels significantly dropped in RSA women compared to healthy fertile women (47). In current study, results of C4 levels agreed with Khayatet al. while C3 levels did not.The present study revealed non-significant dropping in C3 levels in RSA women compared to healthy women.

CONCLUSION:

The variation in result of some studies may support the hypothesis that strains of HCMV may vary in tropism, virulence, and pathogenic possibility, which in turn is likely linked to the genetic variability manifested in key genes important for pathogenesis within wild-type CMV strains (48).Significantly elevation of proinflammatory cytokines may be due to persistence of CMV infection, while non-significant differences in adaptive immunity cytokines may refer to resolution of such immune response following abortion. Modulation of uterusmilieu of infected gestated women by deviation of immune response toward Th2 response may help to prevent of abortion as a results of Th1 immune response.

ACKNOWLEDGMENTS:

I would like thankfulness to the obstetricians and gynecologists and the staff at the Consulting Clinic of Al-Emamain Al-Khadhemain medical city for their aid in collection blood specimens. Also, we would like to render our colleagues in the unit of Medical Research / Faculty of Medicine / Al Nahrain University for their support and assistance in accomplish of current study.

REFERENCES:

1. Wu SJ, Villarreal DO, Shedlock DJ, Weiner DB.Synthetic DNA approach to cytomegalovirus vaccine/immune therapy.Advances in experimental medicine and biology. 2015;848:131-48.

2. Vanarsdall AL, Johnson DC. Human cytomegalovirus entry into cells.Current opinion in virology. 2012;2(1):37-42.

3. Marsico C. and Kimberlin D.W. Congenital Cytomegalovirus infection:advances and challenges in diagnosis, prevention and treatment. Italian Journal of Pediatrics. (2017) 43:38.

4. Manicklal S, Emery VC, Lazzarotto T. et al. The “silent” global burden of congenital cytomegalovirus.ClinMicrobiol Rev. 2013; 26(1): 86–102.

5. Bricker L, Farquharson RG. Types of pregnancy loss in recurrent miscarriage: implications for research and clinical practice. Hum Reprod. 2002; 17:1345–1350.

6. Janeway CA, Travers P, Walport M, Shlomchik M. Immunobiology. 5th ed. New York and London: Garland Science; 2001.

7. Jackson SE, Mason GM, Wills MR. Human cytomegalovirus immunity and immune evasion. Virus Res. 2011; 157:151–160.

8. Amsler L, Verweij MC, DeFilippis VR. The tiers and dimensions of evasion of the type I interferon response by human cytomegalovirus. J Mol Biol. 2013; 425:4857–4871.

9. Fliss PM, Brune W. Prevention of cellular suicide by cytomegaloviruses. Viruses.2012; 4:1928–1949.

10. McSharry BP, Avdic S, Slobedman B. Human cytomegalovirus encoded homologs of cytokines, chemokines and their receptors: roles in immunomodulation. Viruses. 2012;4: 2448–2470.

11. Cheung AK et al. The role of the human cytomegalovirus UL111A gene in down-regulating CD4+ T-cell recognition of latently infected cells: implications for virus elimination during latency. Blood. 2009;114:4128–4137.

12. Stern JL, SlobedmanB. Human cytomegalovirus latent infection of myeloid cells directs monocyte migration by up-regulating monocyte chemotactic protein-1. J Immunol.2008; 180:6577–6585.

13. W. Ouyang, S. Rutz, N. K. Crellin, P. A. Valdez, and S. G. Hymowitz, “Regulation and functions of the IL-10 family of cytokines in inflammation and disease,” Annual Review of Immunology. 2011; vol. 29, pp. 71–109.

14. D. G. Brooks, M. J. Trifilo, K. H. Edelmann, L. Teyton, D. B. McGavern, and M. B. A. Oldstone, “Interleukin-10 determines viral clearance or persistence in vivo,” Nature Medicine. 2006;vol. 12, no. 11, pp. 1301–1309.

15. S. S. Puntambekar, C. C. Bergmann, C. Savarin et al., “Shifting hierarchies of interleukin-10-producing T cell populations in the central nervous system during acute and persistent viral encephalomyelitis,” Journal of Virology. 2011; vol. 85, no. 13, pp. 6702–6713.

16. Rojas JM, Avia M, Martín V, Sevilla N. IL-10: A Multifunctional Cytokine in Viral Infections. Journal of Immunology Research. 2017; 2017:6104054.

17. Shalev I, Schmelzle M, Robson SC, Levy G. Making sense of regulatory T cell suppressive function. SeminImmunol. 2011; 23: 282-292.

18. Hoves S, Krause SW, Schütz C, Halbritter D, Schölmerich J, et al. Monocyte-derived human macrophages mediate anergy in allogeneic T cells and induce regulatory T cells. J Immunol.2006; 177: 2691-2698.

19. Cope A, Le Friec G, Cardone J, Kemper C. The Th1 life cycle: molecular control of IFN-γ to IL-10 switching. Trends Immunol.2011; 32: 278-286.

20. Laouini D, Alenius H, Bryce P, Oettgen H, Tsitsikov E, et al. IL-10 is critical for Th2 responses in a murine model of allergic dermatitis. J Clin Invest. 2003; 112: 1058-1066.

21. 21. Tanaka A, Moriyama M, Nakashima H, Miyake K, Hayashida JN, et al. Th2 and regulatory immune reactions contribute to IgG4 production and the initiation of Mikulicz disease. Arthritis Rheum.2012; 64: 254-263.

22. 22. Joo H, Coquery C, Xue Y, Gayet I, Dillon SR, et al. Serum from patients with SLE instructs monocytes to promote IgG and IgA plasmablast differentiation. J Exp Med. 2012; 209: 1335-1348.

23. 23. Hatachi S, Iwai Y, Kawano S, Morinobu S, Kobayashi M, et al. CD4+ PD-1+ T cells accumulate as unique anergic cells in rheumatoid arthritis synovial fluid. J Rheumatol.2003; 30: 1410-1419.

24. 24. Tang-Feldman YJ, Lochhead GR, Lochhead SR, Yu C, Pomeroy C. Interleukin-10 repletion suppresses pro-inflammatory cytokines and decreases liver pathology without altering viral replication in murine cytomegalovirus (MCMV)-infected IL-10 knockout mice. Inflamm Res. 2011; 60: 233-243.

25. 25. Frossard CP, Eigenmann PA. The role of IL-10 in preventing foodinduced anaphylaxis.Expert OpinBiolTher.2008; 8: 1309-1317.

26. 26. Perricone R, De Carolis C, Giacomelli R, Guarino MD, De Sanctis G, Fontana L. GM-CSF and pregnancy: evidence of significantly reduced blood concentrations in unexplained recurrent abortion efficiently reverted by intravenous immunoglobulin treatment. Am J ReprodImmunol. 2003; Sep;50(3):232-7.

27. Shiomi A, Usui T. Pivotal roles of GM-CSF in autoimmunity andinflammation. Mediators Inflamm. 2015;2015:568543.

28. Hamilton JA. GM-CSF in inflammation and autoimmunity. Trends Immunol.2002;23:403–8.

29. Fleetwood AJ, Lawrence T, Hamilton JA, Cook AD. Granulocyte-macrophage colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes display differences in cytokine profiles and transcription factor activities: implications for CSF blockade in inflammation. J Immunol. 2007;178:5245–52.

30. Al-Dahmoshi, Hussein O.M.; Al-Mammori, Raheem T.O.; ShareefHasanain K.I. , Al-Khafagee Noor S.K. Study of IL-8 and IL-17 levels among certain group of Repeated Spontaneous Abortion Women with or without Toxoplasmosis, Iraq. International Research Journal of Biological Sciences. 2013; vol. 2(8), 37-41.

31. Madhappan B., Kempuraj D., Christodoulou S., Tsapikidis S., Boucher W., Karagannis U., Athanassiou A. and TheoharidesT.C., High levels of Intrauterine Corticotropinreleasinghormone, Urocortin, Tryptase, and Interleukin-8 in spontaneous abortions. Endocrinology, 2003; 144, 2285-2290.

32. Zicari A., Ticconi C., Realacci M., Cela O., Santangelo C., Pietropolli A., Russo M.A. and Piccione E., Hormonal regulation of cytokine release by human fetal membranes at term gestation:effects of oxytocin, hydrocortisone and progesterone on tumor necrosis factor-alpha and transforming growth factor-1 output, J. Reprod. Immunol.2002;56, 123-136.

33. Koumantaki Y., Matalliotakis I.,kyriakou D., Neonaki M.,Goymenon A. and Koumantakis E., Detection of interleukin-6,interleukin-8 and interleukin-11 in plasma from women withspontaneous abortion. Eur. J. Obstet. Gynecol.Reprod. Biol.2001,98, 66-7.

34. Marx L., Arch P., Kieslich C., Mitterlechner S., Kapp M. andDietl J., Decidual mast cells might be involved in the onset ofhuman first-trimester abortion, Am. J. Reprod.Immunol.1999; 41,34-40.

35. Saito S., Miyazaki S. and Sasaki Y.,Thl/Th2 Balance of theimpla-ntation site in humans. Immunology of Pregnancy, 2^ndeds. Edited by Mor G. Eurekah. Com., 1-12 (2004).

36. Alsamarai A. M., Majeed H. M., Alobaidi A. H. Interleukin-6 [Il-6], Interleukin-17 [Il-17] and Angiopoietin in Women with Bad Obstetric History Kirkuk, Iraq. American Research Journal of Hematology. 2016;volume 1, Issue 1, pp: 9 -19.

37. Paradisi, R. Maldini-Casadei, M. Boni, P. Busacchi, P, Porcu, E.Venturoli, S. “T-helper 2-cytokine levels in women with threatenedabortion”. European Journal of Obstetrics Gynecology and Reproductive Biology. 2003;111(1):43–49

38. Von Linsingen, R. Bompeixe, E.P. BicalhoMda, G. “A case-controlstudy in IL6 and TGFB1 gene polymorphisms and recurrentspontaneous abortion in southern Brazilian patients”. AmericanJournal of Reproductive Immunology. 2005;53(2):94–99.

39. Prigoshin, N. Tambutti ,M. Larriba, J. Gogorza, S. Testa ,R. “Cytokinegene polymorphisms in recurrent pregnancy loss of unknowncause”. American Journal of Reproductive Immunology.2004;52(1):36–41.

40. Saijo, Y. Sata, F. Yamada, H. Kondo, T. Kato, E.H. Kishi, R. “Singlenucleotide polymorphisms in the promoter region of theinterleukin-6 gene and the risk of recurrent pregnancy loss in Japanese women”. Fertility and Sterility. 2004;81(2):374–378

41. Lim, K.J. Odukoya, O.A. Ajjan, R.A. Li, T.C. Weetman, A.P. Cooke, I.D. “The role of T-helper cytokines in human reproduction. Fertil.Steril.2000; 73, 136–142.

42. Raghupathy, R. Makhseed, M. Azizieh, F. Hassan, N. Al-Azemi, M. Al- Shamali, E. “Maternal Th1- and Th2-type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortions”. Cell Immunol.1999; 196, 122–130.

43. Calleja-Agius,J. Muttukrishna, S.Arnol,d R. ,Eric Jauniaux,P.”Pro- and antiinflammatory cytokines in threatened miscarriage”s. American Journal Obstetrics and Gynecology. 2011;205(1):83-8.

44. Zenclussen, A.C. Kortebani,G. Mazzolli, A., Margni, R.MalanBorel, I.,” Interleukin-6 and soluble interleukin-6 receptor serum levels in recurrent spontaneous abortion women immunized with paternal white cells”. Am. J. Reprod. Immunol.2000; 44: 22–29.

45. Sharief M., Mohammed R., Shani W.S. The role of Th1 and Th2 cytokines among women with recurrent spontaneous miscarriage. Scientific Journal of Medical Science (2014) 3(7) 345-351. ISSN 2322-5025.doi: 10.14196/sjms.v3.i7.1514.

46. Walia, G.K., Mukhopadhyay, R., Saraswathy, K.N., Puri, M., Chahal, S.M.S.Immuno-Molecular Etiology of Recurrent Pregnancy Loss and the Anthropological Perspective. Int. J. Hum. Genet. 2008; 8 (1-2), 227-235.

47. Zakarea A. Yaseen Al-khayat, NabeelEliaWaheda, Nabaz Faisal Shaker. Complement C3 and C4 Levels in Recurrent Aborting Women with or without Antiphospholipid and Anticardiolipin Autoantibodies.Ibnosina J Med BS. 2014; 143-148.

48. Emery VC and Lazzarotto T. Cytomegalovirus in pregnancy and the neonate [version 1; referees: 2 approved]. F1000Research 2017, 6 (F1000 Faculty Rev):138.

Received on 12.12.2017 Modified on 03.01.2018

Research J. Pharm. and Tech 2018; 11(2):599-603.

DOI: 10.5958/0974-360X.2018.00110.5