Hepcidin mediated iron homoeostasis as immune regulator in visceral leishmaniasis patients

Abstract

Aim: Iron is key ingredient for immunosurveillance and host-pathogen interaction. Intracellular pathogen steals the iron from the host, but how parasite orchestrates iron acquisition and affects immune responses remains controversial. We aimed to study the iron homoeostasis in visceral leishmaniasis (VL) and its influence on immune machinery.

Methods and results: This study was performed on purified monocytes and T cells, peripheral blood mononuclear cells and splenic aspirates for transcriptional analyses of iron homoeostasis (hepcidin, DMT1, transferrin receptor, ferroportin) and immune modulations (IFN-γ, HLA-DR, IL-10, iNOS, IL-6). Serum/plasma was used for determination of iron, total/transferrin iron-binding capacity and anti-leishmania antibody titres in cases. We report that VL-induced perturbation in iron homoeostasis may cause immune dysfunctions. VL cases had decreased iron uptake by transferrin-dependent and transferrin-independent routes while elevated hepcidin, degraded sole iron exporter ferroportin. Therefore, it appears that perturbation in iron homoeostasis has essential role in HLA-DR mediated antigen presentation and innate armoury by downregulating iNOS as well as altering IFN-γ, IL-6 and IL-10 profiles.

Conclusion: The iron homoeostasis by hepcidin can serve as one of the crucial determinants for regulating immune cell signalling; therefore, targeting iron metabolism, specifically hepcidin alone or in combination with agonists, can serve to clear infection.

Keywords: anaemia; antigen presentation; hepcidin; iron homoeostasis; nitric oxide; pro-inflammatory cytokines; visceral leishmaniasis.

Figure 1

Altered clinical profiles in L.donovani infection. (a) hemoglobin levels, (b) WBC count (per microlitre blood sample) in pre-(D-0) and post-treated (DIS) cases, (c) linear regression plot showing strong correlation between WBC counts and hemoglobin levels at pre-treated stage ( p=0.006, Pearson’s coefficient, r=0.2991) (d) serum/plasma transferrin iron binding capacity(TIBC) from pre-treated cases versus healthy controls, (e) linear regression plot showing strong inverse correlation between parasite-specific antibody titres with hemoglobin and (f) linear regression plot showing strong inverse correlation between spleen size(in centimeters) and hemoglobin levels. Comparison between pre (D-0) and post-treatment (D-Dis) were done using paired t-test. Comparison between VL and EHC was done with Mann-Whitney test. (ns=non-significant,**p<0.01, ***p<0.001).

Figure 2

Relative mRNA expression of iron homeostatic transporters. mRNA levels of iron transporters in PBMCs as well as spleen and their comparison; and purified immune cells (CD14+ and CD3+) (a) Transferrin receptor1(TfR1) transcripts of endemic healthy controls(EC), pre- treated (D-0) and post-treated cases(DIS) (after 30 days of treatment), (b) divalent metal transporter-1(DMT-1), (c) hepcidin (HPN) and (d) ferroportin(FPN). Each symbol represents one sample. Comparison between VL and EC were done using t-test and between cell populations originating from the same donors using a paired t-test. Non-parametric tests (Mann–Whitney U test or Wilcoxon matched pair test) were used when normality test failed. (ns=non-significant,**p<0.01, ***p<0.001).

Figure 3

Relative mRNA expression of major effector molecules in leukocytes of VL cases. mRNA expression of (a) HLA-DR in VL (pre- and post-treatment) and EC PBMC as well as splenoctes, and (b) comparative expression levels of iNOS in PBMCs and splenocytes. Comparison between VL and EC were done using t-test and between cell populations originating from the same donors using a paired t-test. Non-parametric tests (Mann–Whitney U test or Wilcoxon matched pair test) were used when normality test failed. (ns=non-significant,**p<0.01, ***p<0.001). (c) linear regression plot for inverse correlation between hepcidin expression and hemoglobin levels ns= non-significant and significant differences are indicated by p-values (* p<0.05, **p<0.01, and ***p<0.001).

Figure 4

Relative expression of effector cytokines in VL cases. (a) Relative mRNA expression of IFN-ϒ in PBMCs of pre- (D-0) and post-treated (DIS) cases as compared to healthy controls (EC) and splenocytes (b) mRNA expression of IL-6 in PBMCs and splenocytes and (c) mRNA expression of IL-10 in PBMCs and splenocytes. Comparison between VL and EC were done using t-test and between cell populations originating from the same donors using a paired t-test. Non-parametric tests (Mann–Whitney U test or Wilcoxon matched pair test) were used when normality test failed. Significant differences are indicated by P-values (* p<0.05, **p<0.01, and ***p<0.001).

Figure 5

Linear regression analysis for correlation of hemoglobin levels with immunological parameters. (a) Inverse correlation observed between hemoglobin levels with IL-10 in PBMCs of pre-(D-0) (p= 0.0105) (b) Similar inverse correlation was observed with the mRNA levels of hepcidin (p= 0.0040) and (c) Direct correlations were observed between the hemoglobin levels with the transcript levels of iNOS(p=0.0257). Pearson’s analysis was performed for the correlational study. Significant differences are indicated by P-values (* p<0.05, **p<0.01, and ***p<0.001).