Twenty-two patients with MHE, showing a PHES of − 4 or lower just before treatment, were treated with rifaximin for 6 months. The PHES and immunophenotype analyses were repeated at 3 and 6 months of treatment.
Patients were considered as “responders” if after treatment with rifaximin they did not present MHE, i.e. the PHES was higher than − 4. If after rifaximin treatment the PHES remained at − 4 or lower, the patients were considered as “non-responders”. As summarized in Table 1, 13 out of the 22 patients (59%) were “responders” and improved the PHES and while 9 (41%) were “non-responders”. Analytical parameters before treatment for responder and non-responder patients did not differ significantly. No significant changes in analytical parameters (haemoglobin bilirubin, albumin, ALT, sodium, creatinine, INR or ammonia) were found after rifaximin treatment (Table 1). The PHES scores obtained by controls and by each group of patients are shown in Fig. 1.
Regarding the clinical evolution of patients during treatment with rifaximin at 3 and 6 months, some patients had decompensation (ascites, bacterial peritonitis, hepatorenal syndrome, acute-on-chronic liver failure, …) but there was no significant association between decompensation and the response to rifaximin neither at 3 nor at 6 months of treatment.
As we have recently shown that appearance of MHE is associated with specific changes in the immunophenotype [11], we assessed whether improvement of MHE by rifaximin is associated with reversal of some of these changes.
As previously reported [11], patients with MHE show an increased percentage of intermediate (CD14++CD16+) pro-inflammatory monocytes and a reduced percentage of classical, non-inflammatory CD14++CD16− monocytes (Fig. 2a, b). Treatment with rifaximin reduced the percentage of intermediate (CD14++CD16+) pro-inflammatory monocytes, reaching normal levels at 6 months in responders (Fig. 2b). This was associated with an increase in classical CD14++CD16− (Fig. 2a) and a decrease in non-classical CD14++CD16++ monocytes (Fig. 2c).
In non-responder patients, rifaximin also reduced the percentage of intermediate pro-inflammatory monocytes, but the reduction was milder than in responders, remaining higher than in controls (Fig. 2b).
Non-responder patients also showed a decrease in non-classical monocytes (Fig. 2c), and a slightly increase in classical monocytes after rifaximin treatment (Fig. 2a).
Appearance of MHE is also associated with an increase in autoreactive CD4+CD28− T lymphocytes (Fig. 3a) and a decrease in no-autoreactive CD4+CD28+ T lymphocytes (Fig. 3b). This is in agreement with the previous report [11]. Treatment with rifaximin strongly reduced autoreactive CD4+CD28− T lymphocytes to nearly normal levels in responders but not in non-responder patients (Fig. 3a). This was associated with an increase in no-autoreactive CD4+CD28+ T lymphocytes in responders, but not in non-responder patients (Fig. 3b). In non-responders there was even an increase of autoreactive and a trend to decrease the percentage of no-autoreactive T lymphocytes at 6 months.
When all patients with MHE are considered as a whole, there is a significant increase in the percentage of CD4+ T lymphocytes expressing the early activation marker CD69 (Fig. 3c). The same activation pattern is observed when responder patients are considered alone. The increased expression of CD69 in CD4+ T lymphocytes is completely reversed by treatment with rifaximin in responder patients (Fig. 3c). In contrast, unexpectedly, non-responder patients did not show increased expression of CD69 in CD4+ T lymphocytes at any time, before or after rifaximin treatment (Fig. 3c). When we examined the activation of autoreactive and no-autoreactive CD4+ T cells, we found the same response as in CD4+ T cells: an increased expression of CD69 in MHE patients as a whole which was due to activation in responder patients, and a reversion by rifaximin treatment, whereas non-responder patients did not show increased expression of CD69 in these cell populations (Fig. 3d, e).
We previously reported that patients with MHE have a more potent immunological response that patients without MHE as reflected in cytokine levels in serum [11]. The increases in IL-6, IL-21, IL-17, IL-18, CCL20, CXCL13, IL-15 and CX3CL1 (fractalkine) were higher than in patients without MHE. In addition, the levels of IL-22 were increased in patients with MHE but not in patients without MHE [11].
We now assessed the effects of rifaximin treatment on the serum levels of these cytokines in responders and non-responder patients (Fig. 4). We observe 3 different types of responses depending on the cytokine analyzed.
For IL-17, CXCL13, CX3CL1 (fractalkine) and IL-22 (Fig. 4a–d), treatment with rifaximin reduces the levels of these cytokines to normal values in responders but not in non-responders. Moreover, in non-responders serum levels of most of these cytokines continue increasing with time (Fig. 4a–d).
A second type of response is observed for IL-6 and CCL20, which are reduced by rifaximin both in responders and non-responders (Fig. 4e, f).
A third type of response, similar to that commented above for expression of CD69 is observed for IL-21, IL-15 and IL-18 which are increased in patients with MHE as a whole or in responder patients before treatment, but are not increased in non-responders at any time, even before treatment. As is the case for CD69, treatment with rifaximin also normalized the levels of IL-21, IL-15 and IL-18 in serum of responder patients (Fig. 4g–i).
We also reported that appearance of MHE is associated with expansion of Th follicular and Th22 CD4+ T lymphocytes subsets, as indicated by the increased expression of the specific transcription factors BCL6 and AHR, respectively [11]. The transcription factors RORC, TBX21 and GATA3, markers of Th17, Th1 and Th2 were not altered in patients with or without MHE. We therefore also assessed the effects of rifaximin treatment on the expression of these transcription factors. Again we observed different types of responses. Treatment with rifaximin reduced the expression of BCL6 to normal levels in responders but not in non-responder patients (Fig. 5a). The expression of AHR was increased in patients with MHE and was reduced to normal values both in responders and non-responders (Fig. 5b). Expression of TBX21 (Fig. 5c) and of GATA3 (Fig. 5d) were not affected by MHE or rifaximin treatment. Finally, RORC was not affected by MHE but was reduced by treatment with rifaximin both in responders and non-responder patients (Fig. 5e).
Appearance of MHE is also associated with increased IgG levels in plasma [11]. As shown in Fig. 6, treatment with rifaximin reduced IgG levels to normal values in responder patients but not in non-responder patients.
It is noteworthy that, for many parameters, the beneficial effects of rifaximin are only partial at 3 months and are higher at 6 months of treatment.