The cells of myeloid origin, mature granulocytes and monocytes, are generally acknowledged with innate functions in pathogen clearance; however, they possess a great capacity to modulate T-cell immunity (Chen and Flies, 2013Chen L. Flies D.B. Molecular mechanisms of T cell co-stimulation and co-inhibition.Nat Rev Immunol. 2013; 13 ([published correction appears in Nat Rev Immunol 2013;13:542]): 227-242Crossref PubMed Scopus (1558) Google Scholar). Alternatively, exposure to weak and sustained proinflammatory mediators from chronic pathological conditions as observed in cancer and autoimmunity may create an imbalance in favor of immature myeloid cells (Veglia et al., 2018Veglia F. Perego M. Gabrilovich D. Myeloid-derived suppressor cells coming of age.Nat Immunol. 2018; 19: 108-119Crossref PubMed Scopus (718) Google Scholar; Zhao et al., 2016Zhao Y. Wu T. Shao S. Shi B. Zhao Y. Phenotype, development, and biological function of myeloid-derived suppressor cells.Oncoimmunology. 2016; 5e1004983Crossref PubMed Scopus (103) Google Scholar). Accordingly, immature myeloid cells’ egress from bone marrow is increased, and these cells accumulate in circulation, secondary immune organs, and sites of inflammation (Bronte et al., 2016Bronte V. Brandau S. Chen S.H. Colombo M.P. Frey A.B. Greten T.F. et al.Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.Nat Commun. 2016; 7: 12150Crossref PubMed Scopus (1266) Google Scholar). T-cell effector functions such as proliferation, cytokine secretion, and migration are hampered on interaction with these immature myeloid cells, which are characterized as myeloid-derived suppressor cells (MDSCs) (Gabrilovich and Nagaraj, 2010Gabrilovich D.I. Nagaraj S. Myeloid-derived-supressor cells as regulators of the immune system.Nat Rev Immunol. 2010; 9: 162-174Crossref Scopus (4418) Google Scholar). In contrast to cancer, MDSCs have been regarded as favorable cells because they can support tolerance mechanisms in autoimmune reactions (Cripps and Gorham, 2011Cripps J.G. Gorham J.D. MDSC in autoimmunity.Int Immunopharmacol. 2011; 11: 789-793Crossref PubMed Scopus (97) Google Scholar). Nevertheless, regulatory activities of MDSCs rely on their immature state, and severity of inflammation in autoimmunity may induce myeloid maturation and provide them with proinflammatory features. We showed that polymorphonuclear (PMN)-MDSCs are highly elevated in pemphigus vulgaris (PV) and bullous pemphigoid (BP) and associate with therapy responses in patients with PV. Peripheral blood samples were freshly collected from newly diagnosed, treatment-naive patients with PV (n = 17), patients with BP (n = 22), and age- and sex-matched healthy volunteers (n = 16). The patients enrolled in this cross-sectional study were followed-up for approximately 3 years. A second sample collection was varied at the end of the consolidation phase in which no new lesions were developed for a minimum of 2 weeks, approximately 80% of the lesions were healed, and which coincided with medication tapering. All the patients with BP and 11 of the 17 patients with PV completed the consolidation phase with systemic and/or topical steroids (prednisolone, 0.5–1.5 mg/kg of body weight per day), with or without adjuvant immunosuppressant drugs (azathioprine, 1.5–2 mg/kg of body weight per day; mycophenolate mofetil, 1 g twice a day; dapsone, 50–100 mg/day; or omalizumab, 300 mg for every 4 weeks). During the follow-up, these patients achieved complete remission. However, 6 of the 17 patients with PV could not achieve complete remission and were clinically improved only after rituximab therapy with or without intravenous immunoglobulin infusion. They were categorized as a conventional treatment recalcitrant-PV (r-PV) group. Institutional approval for the experiments and written informed patient consent were obtained. Demographic and treatment characteristics were recorded together with autoimmune bullous skin disorder intensity score (Daniel et al., 2012Daniel B.S. Hertl M. Werth V.P. Eming R. Murrell D.F. Severity score indexes for blistering diseases.Clin Dermatol. 2012; 30: 108-113Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) (Supplementary Table S1). PMN-MDSCs were identified as a prominent population with CD45+CD125−CD11b+CD14−CD66b+ immunophenotype (Figure 1c). Both percentages (healthy, 3.57 ± 2.01%; PV, 12.42 ± 7.16%; r-PV, 7.54 ± 2.60%; BP, 10.3 ± 4.98%) (Figure 1d) and absolute numbers (healthy, 21 ± 12/μl; PV, 1,129 ± 644/μl; r-PV, 423 ± 117/μl; BP, 957 ± 451/μl) (Figure 1e) of PMN-MDSCs were higher than those of healthy controls but compatible between patients with PV and those with BP. Even though not reaching the level of statistical significance, patients with r-PV tend to have decreased levels of PMN-MDSCs (Figure 1d and e). Particularly, 55.5% and 66.6% of the patients with PV with PMN-MDSC percentages and absolute numbers, respectively, lower than the median values were progressed as r-PV (Figure 1f). Expectedly, PMN-MDSCs from patients with PV and BP hampered T-cell proliferation; both the percentage of proliferating T cells and the rate of proliferation were significantly decreased, and IFN-γ secretion was also decreased in the presence of PMN-MDSCs (Supplementary Figure S1). At the time of admission to the clinic and initial diagnosis, in terms of oral and skin involvement, there was no difference between patients with PV and those with r-PV. Moreover, both the absolute number and percentage of PMN-MDSCs were not significantly correlated with the involvement scores in either PV or r-PV (Figure 2a and b). In patients with BP, the percentage of PMN-MDSCs showed a moderate inverse correlation with the severity of skin involvement (Figure 2c). These cells were not associated with oral involvement in BP (Figure 2d).Figure 2Association of PMN-MDSC levels with clinical features in patients with PV and BP. The correlation analyses between PMN-MDSC percentages and (a, c) skin and (b, d) oral involvement scores of patients with (a, b) PV and (b, c) BP. (e) Kaplan‒Meier curves for progression-free survival of the patients with PV who were divided into two subgroups according to PMN-MDSC percentages higher and lower than the median value 10.1%. (f, h) Percentage and (g, i) absolute counts of (f, g) total neutrophils and (h, i) PMN-MDSCs in patients with PV and r-PV before and after the consolidation phase. (j) Representative flow cytometry dot plots showing the PMN-MDSC population in patients with PV and r-PV before and after treat. BP, bullous pemphigoid; MDSC, myeloid-derived suppressor cell; PMN, polymorphonuclear; PV, pemphigus vulgaris; r-PV, recalcitrant-PV; treat., treatment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) When the total PV cases were distributed into two subgroups according to the PMN-MDSC median percentage (cutoff, 10.16%), the patients with high-level PMN-MDSC had improved post-treatment disease-free survival (Figure 2e). Of note, 55.5% and 11.2% of the patients in the low-level and the high-level PMN-MDSC groups, respectively, were categorized as patients with r-PV. At the end of the consolidation phase, the levels of PMN cells and PMN-MDSCs in the patients were reassessed. The patients with PV who went into remission after their first conventional treatment had pretreatment PMN-MDSC values that approached those of healthy controls (data not shown). A percentage of total PMN cells showed negligible changes, although the number of PMN cells tend to increase in patients with r-PV after the treatment (pretreatment: median = 4.75 × 103/μl, range = 2.5–6.3 × 103/μl; post-treatment: median = 6.3 × 103/μl, range = 3.2–8.8 ×103/μl) (Figure 2f and g). On the other hand, both the percentage and the number of PMN-MDSCs were notably modulated after the therapy. In patients with PV who responded to conventional therapy, PMN-MDSC percentage showed a significant decrease (pretreatment: median 14.7%, range 1.4–20.9%; post-treatment: median 6.08%, range = 3.73–11.12%) (Figure 2h and j). This decrement was also observed in terms of absolute counts (Figure 2i). Intriguingly, after the conventional therapy, PMN-MDSC percentages (pretreatment: median = 7.78%, range = 3.45–10.6%; post-treatment: median = 12.6%, range = 7.87–17.65%) and numbers (pretreatment: median = 384 cells/μl, range = 323–648 cells/μl; post-treatment: median = 972 cells/μl, range = 611–1,355 cells/μl) were remarkably increased in patients with r-PV (Figure 2h–j). In summary, albeit not being related to the severity of clinical symptoms, the amount of circulating PMN-MDSCs in patients with PV is altered according to the course of the disease and response to the therapy. Less number of circulating PMN-MDSCs was commonly observed in a group of patients with PV who were recalcitrant to conventional therapy. Several explanations can be proposed for the low-level PMN-MDSCs in patients with r-PV: (i) inflammatory signals might bear a distinct character that does not induce immature PMN-MDSC production and/or egress from the bone marrow; (ii) in the circulation, inflammatory mediators induce myeloid maturation, which increases in cell density, and PMN-MDSCs become excluded from low-density gradient (the PBMC phase); and (iii) in r-PV, the cells with PMN-MDSC immunophenotype might possess diminished immune regulatory capacities. Further analyses are needed to better characterize the functional properties of the PMN-MDSCs from patients with r-PV. PMN-MDSCs not only interfere with T helper type 1 responses by hampering T-cell proliferation and IFN-γ production but also can hamper B-cell viability, B-cell proliferation, and antibody production (Boros et al., 2016Boros P. Ochando J. Zeher M. Myeloid derived suppressor cells and autoimmunity.Hum Immunol. 2016; 77: 631-636Crossref PubMed Scopus (46) Google Scholar; Lelis et al., 2017Lelis F.J.N. Jaufmann J. Singh A. Fromm K. Teschner A.C. Pöschel S. et al.Myeloid-derived suppressor cells modulate B-cell responses.Immunol Lett. 2017; 188: 108-115Crossref PubMed Scopus (35) Google Scholar). Therefore, both T helper type 1 and B-cell responses can be modulated in the patients with higher PMN-MDSC levels, and these cells may have simply contributed to therapy response. This hypothesis may also explain the therapy resistance in patients with low-level MDSCs. The steroids commonly used in conventional therapy for autoimmune disorders not only suppress the immune responses but also induce the differentiation and production of myeloid cells (Bhattacharjee et al., 2016Bhattacharjee R. De D. Handa S. Minz R.W. Saikia B. Joshi N. Assessment of the effects of rituximab monotherapy on different subsets of circulating T-regulatory cells and clinical disease severity in severe pemphigus vulgaris.Dermatology. 2016; 232: 572-577Crossref PubMed Scopus (14) Google Scholar; Salopek et al., 2002Salopek T.G. Logsetty S. Tredget E.E. Anti-CD20 chimeric monoclonal antibody (rituximab) for the treatment of recalcitrant, life-threatening pemphigus vulgaris with implications in the pathogenesis of the disorder.J Am Acad Dermatol. 2002; 47: 785-788Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar; Schmidt et al., 2019Schmidt E. Kasperkiewicz M. Joly P. Pemphigus.Lancet. 2019; 394: 882-894Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In addition, glucocorticoids, which are a class of corticosteroids, administered in mouse models potentiate the immune-suppressive functions of PMN-MDSCs (Hiçsönmez, 2006Hiçsönmez G. The effect of steroid on myeloid leukemic cells: the potential of short-course high-dose methylprednisolone treatment in inducing differentiation, apoptosis and in stimulating myelopoiesis.Leuk Res. 2006; 30: 60-68Crossref PubMed Scopus (34) Google Scholar; Lu et al., 2018Lu Y. Liu H. Bi Y. Yang H. Li Y. Wang J. et al.Glucocorticoid receptor promotes the function of myeloid-derived suppressor cells by suppressing HIF1α-dependent glycolysis.Cell Mol Immunol. 2018; 15: 618-629Crossref PubMed Scopus (35) Google Scholar). Therefore, the decrease in PMN-MDSC levels in patients with PV observed after the therapy might be directly associated with the mitigation of inflammation, which leads to reduced production of these immature blasts. On the other hand, we were intrigued by the upsurge of PMN-MDSCs after the initial therapy in patients with r-PV. Because they did not achieve complete remission during therapy, inflammatory signals should have been maintained in the patients with r-PV. Together with inflammation, the steroids administered might have augmented the production and egress of PMN-MDSCs from the bone marrow. However, these results may indicate that circulating PMN-MDSC is not sufficient to reestablish the immune tolerance and control the disease progression. In conclusion, PMN-MDSCs were significantly elevated in the circulation of patients with PV and BP and characterized as immune regulatory cells in these autoimmune-blistering disorders. Moreover, PMN-MDSC levels may serve as a predictive value for disease prognosis and therapy responses. The datasets generated and/or analyzed during this study are available from the corresponding author on reasonable request. Ayse Oktem: http://orcid.org/0000-0003-3810-6188 Utku Horzum: http://orcid.org/0000-0002-6747-3043 Pelin Ertop: http://orcid.org/0000-0003-4465-0585 Nihal Kundakci: http://orcid.org/0000-0002-2586-1136 Bengu Nisa Akay: http://orcid.org/0000-0002-4896-1666 Basak Yalcin: http://orcid.org/0000-0003-2297-1409 Günes Esendagli: http://orcid.org/0000-0003-4865-2377 The authors state no conflicts of interest. This work was supported and funded by the Turkish Society of Dermatology. It is covered under the European Cooperation in Science and Technology Action BM1404 (European Network of Investigators Triggering Exploratory Research on Myeloid Regulatory Cells) (http://www.myeeuniter.eu). Cooperation in Science and Technology is supported by the European Framework Program Horizon 2020. Conceptualization: GE, AO; Data Curation: AO, UH; Formal Analysis: UH, AO; Funding Acquisition: AO; Investigation: AO, UH; Methodology: AO, GE, UH; Project Administration: AO; Resources: GE, UH; Software: UH.; Supervision: GE; Validation: AO, UH; Visualization: AO, UH, GE, PE; Writing - Original Draft Preparation: AO, UH, GE; Writing - Review and Editing: AO, UH, GE, PE, BNA, NK, BY Supplementary Table S1Patient and HC DataVariableHCPVr-PVBPNumber (n)1611622Age, y, median (range)44 (28–57)38 (20–66)49 (36–68)79 (45–91)Gender, female/male10/66/55/115/7Oral involvement score, median (range)—5 (2–11)7 (4–10)0 (0–7)Skin involvement score, median (range)—4 (0–15)8.5 (1–92.5)30 (1–135)Time to disease control, median (range)—38 d (10–60 d)Recalcitrant to initial therapy—Abbreviations: BP, bullous pemphigoid; HC, healthy control; PV, pemphigus vulgaris; r-PV, recalcitrant-PV. Open table in a new tab Abbreviations: BP, bullous pemphigoid; HC, healthy control; PV, pemphigus vulgaris; r-PV, recalcitrant-PV.
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