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BCG restores antiviral immune response in patients with Recurrent Respiratory Papilomatosis by affecting dendritic cell differentiation

Evelina Vetskova1*, Toma Avramov2, Maria Muhtarova3, Tzvetelina Stefanova1 and Maria Nikolova3
  • 1 BB-NCIPD Ltd., Bulgaria
  • 2 University Hospital for Active Treatment "Tsaritsa Joanna", Bulgaria
  • 3 National Center of Infectious and Parasitic Diseases (NCIPD), Bulgaria

Recurrent respiratory papillomatosis (RRP) is characterized by recurrent proliferation of benign squamous papillomas within the respiratory tract causing severe airway obstruction (1). The papillomas occur most frequently in the larynx but also – the oropharynx, trachea, bronchi and esophagus (2). Despite its benign nature, in 3-5% of patients, respiratory papillomas undergo malignant transformation to squamous cell carcinoma with quite poor prognosis (3-5). More than 90% of RRP cases are caused by human papillomavirus (HPV) type 6 and 11. Patients infected by HPV-11 develop more aggressive disease (e.g significant airway obstruction) (6). Classical surgery does not prevent relapses, and its combination with antiviral or IFNγ therapy has no significant success.
Efficient control of viral infections requires NK cell activation, differentiation of Th1 and cytotoxic T-cell (Tc1) effectors in combination with regulatory CD4+FoxP3+ T cells with inhibitory function (Treg) to prevent terminal effector cell differentiation and restrict tissue damage caused by non-specific immune activation (7). In addition, plasmocytoid dendritic cells (pDCs) contribute to Th1 differentiation by a massive secretion of IFN-I and provide an essential link between innate and adaptive immune response to chronic viral infections (8). However, persistent viral infections are often associated with depletion or functional impairment of pDCs, misbalanced differentiation of proinflamatory, effector and Treg cells and disrupted Th1/Th2/Th17 cytokine balance (9).
The Bulgarian preparation Calgevax (BulBio-NCIPD Ltd) contains freeze-dried live bacteria derived from a culture of Bacillus of Calmette and Guerin (BCG). It is a potent stimulator of Th1 response and has been successfully applied for treatment of superficial bladder tumors, and malignant melanoma (10). However, data about the effects of combined surgical/ BCG therapy in RRP patients are scarce (11).
The present study investigates the effects of BCG on antiviral immune response in RRP patients subjected to combined CO2 microsurgery /BCG therapy.

Materials and Methods
RRP patients (n = 17) subjected to combined CO2 laser microsurgery / BCG-immunotherapy were studied before (0), 6, 12 and 20 months after the start of immunomodulation. “Calgevax” 2.56 х 10e8 CFU was applied by scarification (on 25 cm² skin area) in 12 applications at 40-45 days’ intervals comprising a period of 18 to 20 months.The control group included age- and sex-matched healthy individuals corresponding to the CLSI standards. Percentages of circulating pDC (LinnegHLA-DR+CD123+CD11clo ), and mature (LinnegHLA-DR+ CD86+) DCs were studied by multicolor flow cytomerty (FACSCanto II, BD). Treg (CD4+ CD25hi+FoxP3+) cells were determined by FoxP3 intracellular staining. Percentage of Th17(CD4+CD69+IL-17+) cells were determined after overnight in vitro PHA stimulation and IL-17 staining. The in vitro PHA-stimulated secretion of IL-2, IL-4, IL-5, IL-10, IFNγ and TNFα was measured by flow cytometry (BDCBAkit). Intragroup and intergroup comparisons were performed with appropriate nonparametric statistical methods and software GraphPad, v.5.

Results
Significantly decreased levels of plasmocytoid and mature DCs were established in untreated RRP patients as compared to healthy controls (average 4.8% vs 8% and 32% vs 60% respectively, p >0.5 for both ). After 12 months of BCG application pDCs and CD86+DCs levels were restored to reference values (8.5% and 55% respectively) (Fig.1 and Fig.2). In addition, 12 months of BCG immunotherapy significantly decreased the share of proinflammatory Th17 (0.4 vs. 0.69, p<0.05) (Fig. 3) and increased the level of circulating Treg (4.6 vs. 7.1, p<0.05). The latter increase was followed by normalization to control levels at the end of the study period(Fig.4). The IFNγ/IL-4, and IFNγ/IL-10 ratios, characterizing the effective antiviral response were significantly decreased at baseline (58 vs. 139 and 15 vs. 26 respectively, p<0.05 for both), but were restored to control levels (159 vs. 139 and 28 vs. 26, respectively, p>0.05 for both) after 20 months of BCG application (Fig.5).

Discussion
Persistent HPV infection and the associated chronic diseases are a global health concern. Immune mechanisms involved in the control of HPV infection, and RRP in particular remain poorly understood. We studied the cellular immune response in RRP patients subjected to combined CO2 microsurgery and BCG immunotherapy in order to pinpoint some mechanisms of its inefficiency and the possibilities for its restoration.
Our results indicate that RRP is associated with depletion of plasmocytoid dendritic cells, misbalanced differentiation of proinflamatory, effector and Treg cells and disrupted Th1/Th2/Th17 cytokine balance. Recent studies have shown that pDC play a pivotal role in anti-viral immunity, mostly by the massive secretion of type I interferons (interferons α and β). It has been established that profound impairment of pDC function during chronic viral infection leads to inefficient T-cell response and maintains virus persistence (8).
As a result of polarization of the immune response in RRP to the Th2/Th17 phenotype, the effective Th1-antiviral immune response to HPV-6 and HPV-11 is disturbed and this imbalance correlates with disease severity (12,13). In RRP Th2 cells dominantly produce IL-4 and IL-10 and inhibit IFN- γ and IL-2 secretion by Th1-cells. This cytokine background (IL-4/IL-10) without IFN-g leads to polarization of naive T cells to regulatory T-cells (CD4+ CD25hi+FoxP3+). However, this leads to inhibition of HPV-specific Th1-immune response (14,15).
The role of Th17 cells during persistent viral infection and in the pathogenesis of chronic viral infection-associated diseases remain unknown. It is considered that Th17 cells and their cytokine IL-17 are strong promoters of persistent viral infection by inhibition of the cytotoxic CD8+ T lymphocytes and of virus-induced apoptosis of infected cells (16). Our results suggest that BCG potentiates the differentiation of Treg at the expense of proinflammatory Th17 cells and stimulates Th1 cytokines secretion (Fig.6).

Conclusion
BCG increases the efficiency of antiviral T-cell response by stimulating the maturation and differentiation of pDCs, resulting in efficient antigen presentation, restoration of Th1/Th2/Th17 disbalance, and induction of Treg preventing the terminal differentiation of T cell clones in the settings of chronic infection.

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References

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Keywords: BCG, RRP, HPV, T-cell response, pDCs

Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.

Presentation Type: Abstract

Topic: Adaptive Immunity

Citation: Vetskova E, Avramov T, Muhtarova M, Stefanova T and Nikolova M (2013). BCG restores antiviral immune response in patients with Recurrent Respiratory Papilomatosis by affecting dendritic cell differentiation. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00998

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Received: 28 Jun 2013; Published Online: 22 Aug 2013.

* Correspondence: Ms. Evelina Vetskova, BB-NCIPD Ltd., Sofia, Bulgaria, e.vezer@gmail.com