Whole blood transcriptome analysis in onchocerciasis.

Identifying the molecular mechanisms controlling the host's response to infection with Onchocerca volvulus is important to understand how the human host controls such parasitic infection. Little is known of the cellular immune response upon infection with O. volvulus. We performed a transcriptomic study using PAXgene-preserved whole blood from 30 nodule-positive individuals and 21 non-endemic controls. It was found that of the 45,042 transcripts that were mapped to the human genome, 544 were found to be upregulated and 447 to be downregulated in nodule-positive individuals (adjusted P-value < 0.05). Pathway analysis was performed on this set of differentially expressed genes, which demonstrated an impact on oxidative phosphorylation and protein translation. Upstream regulator analysis showed that the mTOR associated protein RICTOR appears to play an important role in inducing the transcriptional changes in infected individuals. Functional analysis of the genes affected by infection indicated a suppression of antibody response, Th17 immune response and proliferation of activated T lymphocytes. Multiple regression models were used to select 22 genes that could contribute significantly in the generation of a classifier to predict infection with O. volvulus. For these 22 genes, as well as for 8 reference target genes, validated RT-qPCR assays were developed and used to re-analyze the discovery sample set. These data were used to perform elastic net regularized logistic regression and a panel of 7 genes was found to be the best performing classifier. The resulting algorithm returns a value between 0 and 1, reflecting the predicted probability of being infected. A validation panel of 69 nodule-positive individuals and 5 non-endemic controls was used to validate the performance of this classifier. Based on this validation set only, a sensitivity of 94.2% and a specificity of 60.0% was obtained. When combining the discovery test set and validation set, a sensitivity of 96.0% and a specificity of 92.3% was obtained. Large-scale validation approaches will be necessary to define the intended use for this classifier. Besides the use as marker for infection in MDA efficacy surveys and epidemiological transmission studies, this classifier might also hold potential as pharmacodynamic marker in macrofilaricide clinical trials.

Pathological manifestations in lymphatic filariasis correlate with lack of inhibitory properties of IgG4 antibodies on IgE-activated granulocytes.

Helminth parasites are known to be efficient modulators of their host's immune system. To guarantee their own survival, they induce alongside the classical Th2 a strong regulatory response with high levels of anti-inflammatory cytokines and elevated plasma levels of IgG4. This particular antibody was shown in different models to exhibit immunosuppressive properties. How IgG4 affects the etiopathology of lymphatic filariasis (LF) is however not well characterized. Here we investigate the impact of plasma and affinity-purified IgG/IgG4 fractions from endemic normals (EN) and LF infected pathology patients (CP), asymptomatic microfilaraemic (Mf+) and amicrofilaraemic (Mf-) individuals on IgE/IL3 activated granulocytes. The activation and degranulation states were investigated by monitoring the expression of CD63/HLADR and the release of granule contents (neutrophil elastase (NE), eosinophil cationic protein (ECP) and histamine) respectively by flow cytometry and ELISA. We could show that the activation of granulocytes was inhibited in the presence of plasma from EN and Mf+ individuals whereas those of Mf- and CP presented no effect. This inhibitory capacity was impaired upon depletion of IgG in Mf+ individuals but persisted in IgG-depleted plasma from EN, where it strongly correlated with the expression of IgA. In addition, IgA-depleted fractions failed to suppress granulocyte activation. Strikingly, affinity-purified IgG4 antibodies from EN, Mf+ and Mf- individuals bound granulocytes and inhibited activation and the release of ECP, NE and histamine. In contrast, IgG4 from CP could not bind granulocytes and presented no suppressive capacity. Reduction of both the affinity to, and the suppressive properties of anti-inflammatory IgG4 on granulocytes was reached only when FcγRI and II were blocked simultaneously. These data indicate that IgG4 antibodies from Mf+, Mf- and EN, in contrast to those of CP, natively exhibit FcγRI/II-dependent suppressive properties on granulocytes. Our findings suggest that quantitative and qualitative alterations in IgG4 molecules are associated with the different clinical phenotypes in LF endemic regions.

Hyperreactive onchocerciasis is characterized by a combination of Th17-Th2 immune responses and reduced regulatory T cells.

Clinical manifestations in onchocerciasis range from generalized onchocerciasis (GEO) to the rare but severe hyperreactive (HO)/sowda form. Since disease pathogenesis is associated with host inflammatory reactions, we investigated whether Th17 responses could be related to aggravated pathology in HO. Using flow cytometry, filarial-specific cytokine responses and PCR arrays, we compared the immune cell profiles, including Th subsets, in individuals presenting the two polar forms of infection and endemic normals (EN). In addition to elevated frequencies of memory CD4+ T cells, individuals with HO showed accentuated Th17 and Th2 profiles but decreased CD4+CD25hiFoxp3+ regulatory T cells. These profiles included increased IL-17A+, IL-4+, RORC2+ and GATA3+CD4+ T cell populations. Flow cytometry data was further confirmed using a PCR array since Th17-related genes (IL-17 family members, IL-6, IL-1ß and IL-22) and Th2-related (IL-4, IL-13, STAT6) genes were all significantly up-regulated in HO individuals. In addition, stronger Onchocerca volvulus-specific Th2 responses, especially IL-13, were observed in vitro in hyperreactive individuals when compared to GEO or EN groups. This study provides initial evidence that elevated frequencies of Th17 and Th2 cells form part of the immune network instigating the development of severe onchocerciasis.