Anemia is a condition in which the haemoglobin concentration is lower than normal and might be macrocytic or microcytic. Pernicious anemia (PA) is a megaloblastic anemia consisting of hematological, gastric and immunological alterations. It is an autoimmune disease with a multifactorial etiology involving genetic susceptibility, environmental and immunological factors. Estimate of PA prevalence is 0.1% in the general population which increases to 1.9% in patients aged older than 60 years (1). In older adults of European and African ancestry it is more common (4.0% and 4.3%, respectively) than in people of Asian descent (0.43%) (2, 3).

The immunopathogenesis of pernicious anemia is sustained by autoreactive T cells and by autoantibodies, e.g. intrinsic factor antibodies (IFA) and parietal cell antibodies (PCA). Autoimmunity begins with the activation of gastric dendritic cells, which in turn trigger CD4⁺ T cell lymphocytes in perigastric lymph nodes (4, 5). These autoreactive CD4⁺ T cells as well as the autoantibodies produced by autoreactive B cells are specific for intrinsic factor and the proton pump ATPase, thus attack gastric parietal cells and ultimately lead to gastric cell death and gastric atrophy (6–8). The gastric mucosa atrophy impairs absorption of dietary cobalamin resulting in vitamin B₁₂ deficiency.

Patients develop unexplained fatigue, pallor, cardio-respiratory symptoms like shortness of breath and tachycardia, gastrointestinal manifestations and neurologic abnormalities such as memory loss, poor concentration and paresthesia. It often occurs in combination with other autoimmune disorders such as autoimmune thyroiditis, Addison’s disease, vitiligo, and type 1 diabetes (9, 10).

PA diagnosis includes the serum B₁₂ assay to establish cobalamin deficiency (B₁₂<200ng/L with normal or reduced folate levels) and the detection of serum IFA or PCA (11).

On the other hand, iron deficiency anemia (IDA) is the most common cause of anemia worldwide affecting >12% of the world’s population, especially women, children, and individuals living in under-resourced and middle-income countries. Diagnosis of IDA is based on medical history, microcytic anemia, and a serum ferritin level <15 mg/L (12).

The interleukin (IL)-20 family members, IL-19, IL-20, IL-22, and IL-26 are grouped into the same family based on shared common receptor subunits (the IL-20 receptor β chain, IL-20Rβ) and signaling pathways. All IL-20 subfamily members activate the Janus Kinase (JAK) and signal transducer and activator of transcription (STAT) pathway, particularly STAT3 (13, 14). These subfamily members are part of the larger IL-10 family of cytokines, which besides IL-10 itself, includes the more distantly related IL-28A and IL-29 (which are also known as IFNλ2 and IFNλ1, respectively) (15). The interleukin−20 (IL−20) cytokine subfamily constitutes a key link between the immune system and epithelial tissues, and exerts essential functions in innate mucosal immunity (16). IL-28A and IL-29 are able to induce antiviral responses similar to those of type I interferons and mainly act on epithelial cells (17).

IL-19, IL-20, IL-22, IL-26, IL-28A and IL-29 are produced by different inflammatory cells, primarily keratinocytes, osteocytes, epithelial cells, myeloid cells, B and T cells (14, 18, 19). IL-19 expression can be induced by different stimuli such as TNF-α and IL-17 (20).

IL-19 functions as an anti-inflammatory and pro-angiogenic factor (21). IL-19 was discovered in 2000 and has been implicated in different diseases, such as psoriasis, type I diabetes, endotoxic shock, periodontal disease, vascular diseases and rheumatoid arthritis (22, 23). Xiao et al. recently identified IL-19 as a potent cytokine able to promote the expansion and proliferation of neutrophils. The administration of IL-19 protected wild-type mice from neutropenia induced by both chemotherapy and radiation, demonstrating the therapeutic efficacy of this cytokine. It still remains unknown whether IL-19 has additional effects on other organs or tissues but its potential application in neutropenic state is undoubted, being more efficient than granulocyte colony-stimulating factor (G-CSF). Cancer patients who underwent chemotherapy and had reduced activity to promote bone marrow neutrophil formation, had striking downregulation of serum IL-19 (24).

We wondered whether levels of IL-19 and other related cytokines could be abnormal in patients with anemia, such as PA or IDA. We thus measured serum IL-19, IL-20, IL-26, IL-28A and IL-29 levels in patients with PA, in patients with IDA and in healthy subjects (HC). We then investigated the in vivo production of IL-19 by lamina propria mononuclear cells (LPMC) derived from the stomach of patients with PA and controls.

The present study revealed several major findings that are relevant to the management and understanding the immunological basis of pernicious anemia and autoimmune gastritis.

We investigated the serum levels of different cytokines, such as IL-19, IL-20, IL-26, IL-28A and IL-29 in patients with pernicious anemia and autoimmune gastritis or iron deficient anemia without autoimmune gastritis. No differences in IL-20, IL-26, IL-28A and IL-29 serum levels were present among untreated PA patients, IDA patients or HC. On the other hand, significantly higher levels of IL-19 were found in the sera of PA patients compared to IDA or HC. It is of note that PA is a disease with a clear autoimmune pathogenesis, whose immunopathological features are gastric autoreactive T cells and autoreactive B cells specific for proton pump ATPase and IF, whereas the patients with IDA studied in this research had no autoimmune disease (29, 30). Our results support the notion that IL-19 is a relevant cytokine for the immunopathogenesis of PA but not of IDA.

We can hypothesize that the high serum level of IL-19 found in PA patients might be inversely related to the serum levels of vitamin B₁₂ and that the substitution therapy with B₁₂ will lead to both normal serum levels of B₁₂ and IL-19. It can be further speculated that therapeutic regimen able to reduce IL-19 levels, such as monoclonal antibodies or siRNA, might be useful for the treatment of PA. Given that this investigation represents a pilot single-center study, future multi-center studies dealing with serum IL-19 levels would be very useful for a better definition of the test in PA and IDA.

To investigate whether at gastric level there was any IL-19 production in vivo, we analyzed the IL-19 cytokine production of LPMC derived from the gastric mucosa of patients with PA and HC. We found that LPMC obtained from all PA patients, but not HC, were able to produce high levels of IL-19. It was recently reported in patients with PA that gastric T cells specific for intrinsic factor produced very high level of IL-17 and TNF-α (8). It is of high interest that IL-19 expression can be induced by IL-17, TNF-α and lipopolysaccharide. Moreover, epithelial cells produce IL-19 following stimulation with IL-17, IL-22 and IL-1β (20). In PA patients a different IL-19 cytokine production might be related not only to the local inflammation but also to a different bone stimulation or to other yet unknown interfering factors in IL-19 cytokine production.

Altogether, the results obtained so far indicate that IL-19 serum levels are significantly increased in patients with PA and that IL-19 is produced in vivo in the stomach of PA patients. Thus, we suggest that serum levels of IL-19 may represent a novel important tool to discriminate PA with autoimmune gastritis from IDA without autoimmune gastritis and that the measurement of serum IL-19 might be useful for monitoring the response to B₁₂ vitamin therapy in PA patients.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Ethical Committee Area Vasta Centro Firenze. The patients/participants provided their written informed consent to participate in this study.

CDB, AAn, NB and MMD'E conceived and designed the study with MMD'E supervision. CDB, MPP, NC, MB, LP, AAz, SP, SD'E, FC, DO-P performed the experiments; analysis and interpretation of data were conducted by CDB, AAn, NB, MMD'E. CDB, AAn, NB, MMD'E wrote, reviewed and edited the manuscript. All the authors contributed to the article and approved the submitted version.

This work was supported by the Italian Ministry of University (grant 2010P3S8BR_005).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.