Cytokine and Chemokines Alterations in the Endemic Form of Pemphigus Foliaceus (Fogo Selvagem)

Introduction The endemic form (fogo selvagem—FS) of pemphigus foliaceus is an autoimmune disease characterized by the presence of IgG autoantibodies against desmoglein-1. Despite the array of findings, the role of chemokines and cytokines that dictate the immune response and disease outcome is still poorly investigated. Materials and methods Serum from 64 patients diagnosed with FS was used to draw and establish the levels of these molecules on this disease and establish the levels of these molecules with the severity of FS, and influence of treatment. Results In comparison to healthy subjects, FS patients, newly diagnosed and still without therapeutic intervention, had higher levels of IL-22 and CXCL-8, and reduced levels of IFN-γ, IL-2, IL-15, and CCL-11. Furthermore, treatment using immunosuppressant drugs augmented the production of IFN-γ, IL-2, CCL-5, and CCL-11 besides reducing the levels of IL-22 and CXCL-10. Immunosuppressive therapy seemed to have long-lasting effects on the production of higher amounts of IFN-γ, IL-2, and CCL-5, besides keeping lowered the levels of IL-22 in remission FS patients. Conclusion Taken together, our findings suggest a putative role of IL-22 in the pathogenesis of FS. Finally, data presented here may contribute for better understanding the immune aspects that control disease outcome.

29 Black (n) 6 15 housing area Urban area (n) 18 26 0.55 c Urban and rural areas (n) 15 33 Rural area (n) 4  Fogo selvagem shares clinical and immunopathological features to the non-endemic form and is characterized by the presence of pathogenic autoantibodies (primarily IgG4) against the desmoglein-1 (Dsg1), resulting in loss of organization between keratinocytes (acantholysis) that leads to the formation of intraepidermal vesicles (1,5,6). Despite the role of antibodies targeting desmoglein, several other aspects are associated with the complex pathogenesis and susceptibility of pemphigus. Increased levels of T helper-2 (Th2) cytokines such as IL-4, IL-10, and IL-13 were already shown to be involved in the production of IgG4 by B-lymphocytes in both PF and PV patients. Those patients also exhibited reduced levels of IL-2 and IFN-γ, resulting in suppressed expansion of Th1 lymphocytes, which suggests an inhibitory effect of Th2-cytokines, thus contributing to disease worsening (7).
Owing to the immune imbalance, pharmacological treatment of pemphigus, especially in underdeveloped and developing countries, is mainly based on immune modulatory and anti-inflammatory drugs such as glucocorticoids (GC). However, some patients are refractory or may present some side effects (8). This poor responsiveness could be related to elevated levels of the inflammatory cytokines IL-6 and TNF-α, which can induce GC resistance observed in peripheral blood mononuclear cells (PBMCs) isolated from pemphigus subjects (8). Furthermore, augmented levels of IL-10 and IL-12 concomitant to reduced levels of IL-2, IL-4, IL-5, and IFN-γ were found in PF patients without treatment, when compared to their control counterparts (9). On the other hand, PBMCs isolated from PF patients treated with GC, showed increased levels of both the inflammatory cytokine IL-1β or in the IL-5/IFN-γ, when compared to their healthy counterparts (10). Taken together, the few studies conducted so far only describe cytokines related to Th1 and Th2 pattern of immune response. In other words, these findings do not permit a clear definition of the entire immune aspects related to the onset, treatment, and remission of the disease.
In the last years, other cytokines such as IL-9, IL-17, and IL-22 have been implicated in the pathogenesis of inflammatory and autoimmune diseases affecting skin, such as psoriasis, for example (11,12). IL-9 seems to act synergistically with IL-17 and IL-22, which can be related to disease worsening (12). Though skin diseases such as psoriasis can be associated with the development of pemphigus (13,14), the latter still requires more clarification regarding cytokine interplay, immune profile, and the impact of treatment on disease outcome.
Thus, given the lack of data concerning the immune profile of FS patients in different clinical stages of disease as well as the impact of treatment in this context, this study aimed to elucidate the cytokine/chemokine interplay in those subjects.

Patients
The study was conducted with patients from a hospital for blistering diseases located in Uberaba, state of Minas Gerais, Brazil, from January 2013 to December 2015. Serum from 64 patients diagnosed with FS was used in this study (Table 1; Figure 1). Samples were collected during the consultation of patients in different stages of treatment (before, during, and after treatment) in the hospital for blistering disease. For all patients, peripheral blood samples were collected by venipuncture in an EDTA-coated vacutainer tube (BD Biosciences, San Jose, CA, USA) and kept in ice until plasma separation (within 30 min). Blood was centrifuged at 1,000 × g for 10 min and plasma was aliquoted (0.5 ml per tube) and stored at 80°C until analysis. Among them, 9 did not receive treatment by the time they were diagnosed and samples were collected (untreated subjects), 45 were under treatment with immunosuppressant drugs (treated subjects) ( Table S1 in Supplementary Material), and 10 remissive subjects were treated at least 1 year before samples were collected, did not display any features of disease activity nor were taking any further medication (post-treatment subjects). Treated subjects (n = 45) were further subcategorized in three groups (group I-29, II-6, and III-10 patients) according to the level of cutaneous lesions. The extension of cutaneous damage was assessed according to the Wallace's rule of nines (15). 39 subjects, age-matched, originated from the same areas without diagnosis of autoimmune, inflammatory or infectious diseases, neoplastic disorders, or allergies, composed the control group. Furthermore, none of the individuals from the control group had any familial case of pemphigus. Patients diagnosed with FS had clinical, pathological, and/or serological diagnosis. Informed consent was obtained for experimentation with all human subjects. The median age of FS patients in our study was 36 years old with no predominance of sex. The majority of patients were white or mulattoes (76.6%). The number of patients from urban area (40.6%) or from urban area with frequent contact with rural areas (51.6%) was higher than those living in rural area (7.8%) ( Table 1).
All procedures were conducted in accordance with the Declaration of Helsinki, with full patient compliance and approved human practices as defined by the Ethics Committee for Human Experimentation of the Federal University of Triângulo Mineiro (UFTM), Uberaba, Minas Gerais, Brazil, protocol number 1.311.730.  Figure 2Q). The following cytokines/chemokines were not detected: IL-13, TGF-β, IL-4, IL-9, and CCL3 (data not shown). These results suggest a complex interplay between cytokines and chemokines in the pathogenesis of FS.

The relationship between cytokines, chemokines, and Disease severity
To check for a relationship between disease severity and the levels of cytokines and chemokines, FS patients were stratified according to the extension of lesion ( Figure 3A). The greater severity of disease, which means patients with more than 90% of compromised skin (group III), was associated with higher levels of IFN-γ ( Figure 3B), IL-2 ( Figure 3C), and CCL-5 ( Figure 3H), when compared to the untreated patients. Furthermore, higher levels of IL-22 ( Figure 3G) were also verified in group III, when compared to group I, and in untreated patients compared to group I. The greater severity of disease was also related to higher levels of IL-10

Data analysis and statistics
For all variables, normal distribution and homogeneous variance were tested. The D'Agostino-Pearson test was used to assess normality for all variables. In cases of non-Gaussian distribution of data, the non-parametric Mann-Whitney test was applied. Multiple comparisons relating to the medians of values for more than two groups were made using the Kruskal-Wallis non-parametric test followed by Dunn's test. For correlations the Spearman test was used. The observed differences were considered significant when p < 0.05 (5%). Statistical analysis was performed using the Graphpad Prism software (GraphPad Software 6.0, La Jolla, CA, USA).

resUlTs cytokine and chemokine Profile in Treated and non-Treated subjects
Because of the importance of cytokines and chemokines on different physiological and pathological aspects, and due to the  when compared to group II and untreated patients ( Figure 3F).

cytokine and chemokine interplay after Treatment with immunosuppressant Drugs
Next, to verify if immunosuppressant therapy may induce longlasting effects on cytokine and chemokine levels, we evaluated the cytokine and chemokine levels in remissive patients and compared it with FS subjects before and during treatment with immunosuppressive therapy. Treatment induced and maintained the production of IFN-γ and IL-2 (Figures 4A,H, respectively). However, though the production of IL-33 (Figures 4L,R) and CCL-11 (Figures 4P,R) was induced by treatment, the higher levels of these molecules were not kept after withdrawal of therapy. The levels of the chemokine CCL-5 ( Figure 4O) were higher in FS patients after treatment. The absence of treatment in FS patients was especially associated with higher levels of IL-22 (Figures 4G,R). No differences were observed between the conditions of treatment for the production of IL-17 (Figure 4B), IL-6 ( Figure 4C), IL-23 (Figure 4D), IL-5 (Figure 4E), IL-10 ( Figure 4F), TNF-α (Figure 4I), IL-12 (Figure 4J), IL-15 ( Figure 4K), CXCL-8 (Figure 4M), CCL-2 ( Figure 4N), and CCL-10/IP-10 (Figures 4Q,R). The following cytokines/chemokines were not detected: IL-13, TGF-β, IL-4, IL-9, and CCL3 (data not shown). These results suggest that immunosuppressant therapy may induce long-lasting effects on the production of cytokines and chemokines.

DiscUssiOn
In recent years, some efforts have been made to elucidate the role of cytokines and chemokines in the pathogenesis of FS; however, the findings described so far are limited, and their implications in disease outcome were not fully elucidated yet. In our study, the plasma levels of over 17 cytokines and 6 chemokines were evaluated in FS patients. The results pointed to a putative role of IL-22 in the pathogenesis of FS, and to a supposed protective function of especially Th1-related cytokines and Th2 chemokines in serum. The most affected regions in Brazil include the mid-western and the southeastern states, including Minas Gerais (3,16), where the majority of patients analyzed in our study came from. Furthermore, FS has been primarily described as predominant over people from rural areas, being uncommonly described in urban areas (3). Since its first description, the etiology of FS has been mainly attributed to environmental triggers present in rural areas, which can be different based on geographical distribution and genetic inheritance (17,18). Despite the description of FS as primarily found in rural areas, our study suggested an urbanization of this disease. This scenario could be at least partly attributed to the presence of strong environmental triggers in urban areas, including stress, lifestyle, and pollution, which were already described as potential triggers for other autoimmune diseases such as those affecting thyroid (19), systemic lupus erythematosus, and type 1 diabetes (20), for example. Additionally, the saliva of sand flies contains some components like LJM11, which is a salivary protein from the sand fly Lutzomyia longipalpis (21), able to cross-react to anti-Dsg1 monoclonal autoantibodies from FS patients. This phenomenon, in turn, induced a production of autoantibodies targeting Dsg1 in mice previously immunized with the salivary protein (1,21,22). Accordingly, the so-called "urbanization" of FS observed in our study, can be also attributed to changes in the behavior of the insects moving from sylvatic areas to urban areas as a consequence of social-environmental alterations (23,24). Therefore, it is possible to consider the role of insect-derived saliva proteins, like LJM11, in the onset of a pathogenic response, leading to FS.
The complex interplay between cytokines and chemokines that dictates disease outcome in FS is still a matter of investigation. In our study, the comparison between healthy subjects and FS patients suggested a role of IL-22, and to a less extent IL-8, in the FS. Healthy subjects had higher levels of IFN-γ and IL-2. Similarly, elevated levels of IFN-γ and IL-2, along with a reduction in IL-22, were observed in FS patients under treatment or after treatment withdrawn, when compared to untreated patients. A different study, also pointed to a reduction in serum  of IL-2 and IFN-γ in FS patients when compared to their healthy control counterparts (9). Though in a different pemphigus entity, PV-treated patients also showed elevated levels of IFN-γ (25). Additionally, the inverse relationship between IL-22 and IFN-γ was described in patients with active psoriasis (26). IL-22 is especially produced by Th17 and Th22 cells (27) and is involved in several processes including the regulation of tissue cells in skin, kidney, pancreas, small intestine, liver, colon, and respiratory system (26). Despite the beneficial role of IL-22 in the aforementioned tissues, this cytokine, depending on the context, may present a pro-inflammatory role. For this reason, it has been implicated in the pathogenesis of different inflammatory skin disorders including atopic dermatitis (28), psoriasis (29) and systemic lupus erythematosus with skin manifestations (30). However, the participation of IL-22 in the pathogenesis of FS is still not elucidated and needs to be further explored. In skin, despite the absence of effect over skin fibroblasts, endothelial cells, and melanocytes, proliferation of keratinocytes, epidermal hyperplasia, and abrogated keratinocyte terminal differentiation are mediated by IL-22, mainly derived from Th22 cells (31). IL-22 has shown in vitro, great ability to downregulate key genes in keratinocyte differentiation, including loricrin, filaggrin, and involucrin, which resulted in striking acanthosis (32,33). Because of the effect of IL-22 in skin, higher levels in both skin and serum have been related to disease worsening in patients with active psoriasis (34,35). Even though psoriasis and PF are considered to be distinct entities, in some cases, they can occur together in the same patient. In these cases, PF has been developed in untreated or chronic psoriatic patients (36)(37)(38)(39)(40)(41)(42). However, we cannot exclude the participation of genetics in this context, the presence of HLA DRB1 alleles, which have been described to be related with both psoriasis (43) and PF/FS (44,45), could possibly explain the concomitant occurrence of both diseases. Although we have not determined the presence of the HLA DRB1 alleles in our study, the importance of IL-22 in the pathogenesis of FS cannot be underestimated and needs to be further explored. Additionally, we cannot underestimate the importance of the balance between IL-22, IL-2, and IFN-γ, neither the role of IL-22, on disease onset and progression. As we have pointed out here, in treated patients (even after treatment withdrawn), the opposite relationship between the higher levels of IL-2 and IFN-γ and lower levels of IL-22, appeared to be related to disease amelioration. We believe that in FS, IL-2 and IFN-γ may exert a protective role antagonizing the effects of IL-22.
Despite the crucial role of cytokines in the pathogenesis of FS, chemokines are also important players in this scenario. Our results pointed to higher levels of CXCL-8/IL-8 in FS patients. IL-8 is a chemokine produced during inflammatory stimulation, and its main function is to chemoatract and activate neutrophils (46). Though this chemokine is widely studied owing to its role in neutrophil recruitment and inflammation, data concerning the participation of IL-8 in the pathogenesis of FS still needs to be clarified. In a rare subset of pemphigus, known as pemphigus herpetiformis (PH), in which IgG autoantibodies targeting Dsg1 seems to be related to the development of disease, it was suggested that these autoantibodies were able to induce overexpression of IL-8 in keratinocytes (47). Cultured keratinocytes from these patients showed increased production and secretion of cytoplasmic IL-8 after stimulation with autoantibodies derived from PH patients (47), which reinforces the effect of autoantibodies targeting Dsg1 on the production of IL-8. Similarly, the blister formation in bullous pemphigoid was associated with higher levels in serum of IL-6 and IL-8 (48,49). In contrast, in other subsets of pemphigus, pemphigus vulgaris, higher levels of IL-8 were observed in PV-treated patients (25). Although we have not investigated the relationship between the effects of autoantibodies targeting Dsg1 on the production of IL-8 in our study, we cannot exclude this possibility. Moreover, though not as important as IL-22, IL-8 seems also to be related to disease activity, and together with the former, may represent a potential target for new therapeutic approaches. However, such role of IL-22 and IL-8 in FS pathogenesis needs to be further clarified.
In our study, CCL-5/RANTES was induced and maintained by immunosuppressant drugs even after treatment withdrawn. However, its role in the development of FS is not fully explored. CCL-5 is a chemokine with strong capacity to recruit monocytes, T cells, and eosinophils, acting via the receptors CCR1, CCR3, and CCR5 (50). Its expression in other forms of pemphigus is controversial. Patients diagnosed with bullous pemphigoid, pemphigus, and dermatitis herpetiformis showed no differences for the presence of IL-8 and RANTES in the skin lesions (51) or in serum (52), when compared to their control counterparts. Interestingly, the higher levels of CCL-5, IFN-γ, and IL-2 found in treated patients in our study were followed by a reduction in IL-22. In patients with lung inflammation, higher levels of IL-22 were shown to be able to suppress IFN-γ-induced secretion of CCL-5 (53). The effects of immunosuppressant drugs like prednisone, one of the most prescribed therapies to treat FS, were shown to be involved in the reduction of IL-22 in patients with systemic lupus erythematosus (54). Therefore, we believe that immunosuppressive therapy influenced this balance, reducing the levels of IL-22, and thus favoring the production of IFN-γderived CCL-5. Nevertheless, the mechanisms behind this modulation were not explored in our study. Additionally, it is difficult to address the consistencies/inconsistencies between different subtypes of pemphigus. This is probably due to the distinctions in etiology, physiopathology, geographical area, incidence, and prevalence observed in each case.
We believe that our study is the most detailed and complete study on the effects of cytokines and chemokines in the pathogenesis of the endemic form (FS) of PF. Our results pointed to a putative role of IL-22 in the pathogenesis of FS, and to the importance of immunosuppressive therapy to reestablish the immune balance in FS patients. Ultimately, we believe that this study may provide a better knowledge of the cytokines and chemokines involved in disease outcome, besides suggesting IL-22 as a hypothetical target to be explored in future studies. Moreover, our study demonstrates, for the first time, the urbanization of the disease. These data are also important because future preventive decisions must take into account the environmental triggers in urban areas that may be related with the onset of the disease.

eThics sTaTeMenT
This study was carried out in strict accordance with the principles and guidelines adopted by the Declaration of Helsinki, with full patient compliance and approved human practices as defined by the Ethics Committee for Human Experimentation of the Federal University of Triângulo Mineiro (UFTM), Uberaba, Minas Gerais, Brazil, protocol number 1.300.898.