The discovery of Wolbachia bacterial endosymbiont in filarial nematodes of medical importance, including the causative agent of River Blindness, and of its crucial role in worm biology and disease pathogenesis has opened the opportu- nity for a safe and effective control of onchocerciasis. However, the employment of anti-wolbachial treatment as a public health tool requires further study and optimisation on pharmacology, field implementation strategies and impact on onchocerciasis control. Moreover, this has also raised important questions about the nature of Wolbachia-nematode interaction and the possible effects of anti-filarial therapy targeting Wolbachia in the context of the host immune re- sponses. The work presented here aimed to explore several of these aspects. Although extensive research is ongoing to optimise regimes of existing anti-
Wolbachia drugs and to identify alternative drugs and combinations more suit- able for Mass Drug Administration (MDA), it has recently been shown in a pilot implementation trial in Cameroon that the delivery of the “gold-standard” mac- rofilaricidal 6 week course of doxycycline therapy as a MDA is feasible, safe and well accepted by communities when delivered with a community-directed strat- egy. The work presented here in chapter 2 evaluated the long-term effectiveness of this intervention followed by standard ivermectin (IVM) MDA treatment by assessing prevalence and burden of infection 4 years after implementation and with ongoing transmission. The results showing a significant reduction in mi- crofilaridermia prevalence and load in people that completed the 6 week course of doxycycline followed by IVM compared to those who received only IVM MDA demonstrated the long term effectiveness of doxycycline MDA and validated the very high compliance rate reported in the implementation trial. These results encourage the introduction of anti-wolbachial therapy in onchocerciasis control programmes where needed and provide important data to address the practical aspects of the implementation of macrofilaricidal drugs.
The host inflammatory response to Onchocerca volvulus microfilariae (mf) and their endosymbiont Wolbachia is at the basis of onchocercal pathology. Through TLR2/6 activation by Wolbachia peptidoglycan-associated lipoprotein,
Wolbachia interacts with the host’s immune cells inducing the production of pro-inflammatory cytokines. This may create, in concert with the anti- inflammatory cytokine environment promoted by the filarial worm, a suitable milieu for the development of a Type-17 immune response. This is characterized by the production of the signature pro-inflammatory cytokine IL-
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17 and the recruitment of neutrophils into tissues, this latter being a feature of onchocerciasis strictly dependent on the presence of Wolbachia. Although some data exists on the development of a Type-17 immune response in lymphatic filariasis and its possible role in the pathogenesis of lymphatic disease, so far no investigation was carried out in onchocerciasis or on the role of Wolbachia in this context. Moreover, no human study so far investigated the relative role of
Wolbachia and the filarial nematode in the induction of pro- and anti- inflammatory cytokines in onchocerciasis infected humans ex vivo. In the work presented here in chapter 3, peripheral blood mononuclear cells (PBMC) from patients with onchocerciasis and control subjects from Ghana were stimulated with filarial extracts and the cytokine response was analysed in the light of presence or absence of Wolbachia in the filarial extracts and patients’ microfilaridermia loads. The results showed a predominant Th2 response in patients compared to controls, while Th1 and Th17 responses were minimal in both groups. Thus, the filarial-specific Type-17 immune response appears to be regulated in the same way of Th1 and Th2 responses, which are heightened in pathology but down-regulated by active infection.
Interestingly, parasite loads showed a different influence on Th2 compared to pro- and anti-inflammatory responses with patients with higher mf loads show- ing depressed Th2 responses but heightened spontaneous production of both pro- and anti-inflammatory cytokines. These results suggest that filarial para- sites are able to promote both pro- and anti-inflammatory responses at the same time. This preliminary study did not show any difference in the ability of
Wolbachia-containing and Wolbachia-depleted filarial extracts to stimulate PBMC. However, practical and time constraints prevented a rigorous optimiza- tion and control of reagents and culture conditions, which should be carried out in further studies of this type. These should take advantage of the availabil- ity of adulticidal therapy with doxycycline to investigate the relative role of Wol- bachia and of different parasite life stages in shaping the immune profile in on- chocercal infection and disease using a rigorous classification of patients’ infec- tion and disease status.
The presence of IL-17 was also investigated ex vivo in onchocercomas from pa- tients treated with doxycycline or placebo, as presented in chapter 5. Using immunohistochemistry (IHC), it was found an abundant IL-17+ cell infiltrate
around and in contact with Wolbachia-containing but not Wolbachia-depleted
O. volvulus adults. Interestingly, Th17 cells were present in this infiltrate in a higher percentage compared to other inflammatory diseases characterised by the production of a Type-17 immune response, such as psoriasis and rheuma-
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toid arthritis, suggesting that these cells may have a role in the localised im- mune response elicited by the worm through its endosymbiont in the nodule. However, the vast majority of the IL-17+ cells in onchocercomas were neutro-
phils, a surprising finding that was investigated in more depth using human neutrophils from healthy donors in vitro. Using IHC, unstimulated neutrophils but not neutrophil precursors resulted IL-17+, with an intracellular signal that
increased in intensity upon stimulation with LPS or synthetic Wolbachia pepty- doglycan-associated lipoprotein (WoLP). However, when confirmation of these results was pursued at the protein and the gene transcription level, results were inconclusive. In particular, IL-17 was detected in human neutrophils only by Western Blot, while IL-17 was undetected using ELISA-based assays and different anti-IL-17 antibody sources. Moreover, the small PBMC contamination of purified human neutrophil preparations prevented a definitive conclusion about IL-17 expression by human neutrophils at the mRNA level using RT-PCR. A preliminary experiment using immunoprecipitation followed by mass spec- trometric analysis of neutrophil samples failed to detect IL-17 in cell lysates, but this result can not be considered conclusive in the light of the negative re- sult obtained also in one of the positive-control samples, thus requiring further optimization. Although inconclusive on IL-17 expression by human neutrophils and on the role of Wolbachia in this response, these results highlight the need for a careful interpretation of published data reporting IL-17 expression by these cells, the vast majority of that being based only on the same IHC tech- nique used in this work. Moreover, they also point out the need for a more stringent neutrophil purification technique in further studies assessing the pro- tein expression by neutrophils using RT-PCR. Finally, IL-17 in neutrophil sam- ples should be found, the source of IL-17 in neutrophils, whether endogenous or exogenous, should be also investigated. However, the characterization of binding specificity of the polyclonal anti-human IL-17A antibody used here de- serves further attention in the view of its possible optimization for use as a neu- trophil-specific marker.
A typical feature of onchocercomas harbouring Wolbachia-containing but not
Wolbachia-devoid Onchocerca nematodes is the presence of a rich neutrophil infiltrate. Neutrophils are also involved in Wolbachia-mediated pathogenesis of onchocercal disease manifestations. However, neutrophils do not appear to be detrimental to living worms, and their role in the filarial nematode parasitism is still not known. The study presented in chapter 4 investigated the interaction between human neutrophils and Wolbachia in vitro. The results demonstrate that WoLP activates human neutrophils, as shown by modulation of the surface