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P085 Crohn’s disease patients under combined therapy with Azathioprine and Infliximab present persistent inflammation together with a counter regulatory response during clinical disease remission

M. Duarte-Silva1,2, R. S. Parra*3, M. R. Feitosa3, O. Féres3, J. J. Ribeiro da Rocha3, C. R. d1, B. Cardoso1

1School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil, Department of Clinical Analysis, Toxicology and Food Science, Ribeirão Preto, SP, Brazil, 2Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil, Department of Immunology and Biochemistry, Ribeirão Preto, Brazil, 3Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil, Surgery and Anatomy, Ribeirão Preto, SP, Brazil

Background

Crohn's disease (CD) is characterised by a chronic dysregulation of the gut mucosal responses. This study aimed to evaluate peripheral blood mononuclear cells (PBMC) phenotype and its responsiveness to the activating stimulus of Crohn's disease patients treated with Infliximab (IFX) combined with Azathioprine (AZA).

Methods

We enrolled 20 healthy controls (HC) and 40 CD patients in clinical remission (25 using IFX and 15 using IFX plus AZA—Ethics Committee approval nº. 2.023.23). Immunophenotyping of PBMC was performed by flow cytometry. Leucocytes were stimulated with anti-CD3/CD28 by 5 days or with LPS by six h. Cytokines (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IFN-γ, and TNF-α) were measured in the culture supernatants and plasma samples by Cytometric Bead Array. LPS was measured in plasma by Enzyme Immunoassay.

Results

Combined AZA+IFX therapy led to decreased NK (CD16+) and B cells compared with HC, in contrast to increased CD14+ monocytes, as well as CD14++CD16+ (intermediary) cells in both IFX and combined groups, indicating a tendency towards an inflammatory response. Besides that, LPS and IL-6 were augmented in all CD’ plasma, suggesting that these patients still present bacteria translocation to circulation and systemic inflammation. Moreover, increased amounts of TNF and IL-17A were detected in the supernatant of stimulated cultures of AZA+IFX patients, compared with HC and IFX,, respectively, though the lower levels of IL-17 were found in IFX-treated patients. Most interestingly, there was a notable augment of Foxp3+ cells in CD despite the treatment, indicating a counter regulatory response to the residual inflammation (Tables 1 and 2).

Table 1. Mean of cells frequency.

HC (%)IFX (%)IFX+AZA (%)p-value
Leucocyte population
CD3-CD16+13.04a8.627.04b0.0160
CD3-CD19+8.93a7.045.73b0.0107
CD4+CD25+FoxP3+22.4941.3640.890.0360
CD14+84.49a87.4888.75b0.0500
CD14++CD16+3.44a8.39b7.01b0.0090

‘a’ and ‘b’ represent statistical differences between the indicated groups.

Table 2. LPS and cytokine dosage.

HC (%)IFX (%)IFX+AZA (%)p-value
Plasma
LPS (EU/ml)0.1324a0.2414b0.2493b<0.0001
IL-6 (pg/ml)0.1633a1.62601.4280b0.0214
Supernatant
TNF (pg/ml)1729.0a1921.03254.0b0.0217
IL-17A (pg/ml)280.2185.1a567.5b0.0247
Proliferation (%)48.1364.5462.380.0984

‘a’ and ‘b’ represent statistical differences between the indicated groups.

Conclusion

Patients in disease clinical remission still present relevant markers of inflammation, in spite of the constrained NK/B lymphocytes and augmented regulatory population induced by AZA and IFX treatment, which have relevant impact in the ongoing CD immune response.

Financial support: FAPESP (2017/08651-1).