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    • In DC from A group only

    2019-05-22

    In DC from A group only few genes appeared to be significantly and highly modulated (10 out of 84). The HIV natural ligands CCL4 and CD4, the HIV induced transcription factor IRF1 as well as the apoptotic genes BAD and CASP8 resulted down-regulated compared to monocytes at all the considered time points (iDC, 4 h-DC, 14 h-DC, 24 h-DC and 48 h-DC). In a similar way, innate immune response genes, namely IL1B, IL10, SELL, TNF and TNFSF10, also appeared to be significantly down-regulated at least at one of the differentiation steps. On the other hand, in glutathione reductase from group B we observed a statistically significant modulation in 25 out of 84 genes in quite all the considered time points. In this group, 22 genes resulted up-regulated, whereas only 3 genes resulted down regulated (Table 3). Genes belonging to the apoptotic as well as to “cell activation by HIV-1” pathways were up-regulated in these DC compared to monocytes. Only 5 genes seemed to be significantly modulated in both groups. IL1B and SELL were commonly down-regulated, whereas BAD, CASP8 and IRF1 were oppositely modulated, being down-regulated in DC from group A and up-regulated in group B (Table 3). This finding was not corroborated by viability assay. Mature dendritic cells (48 h-DC) obtained from all the patients were similarly viable (Group A: 80 ± 7% PI-negative cells; Group B: 75 ± 4% PI-negative cells; p = 0.338), however we cannot exclude that cell death could happen later than 48 h. When looking at innate immune response genes (Fig. 3A), we can observe that the expression of IL1B, IL10, SELL, TNF and TNFSF10 was significantly down-regulated in DC belonging to group A. However IL1B and TNF were less down-regulated in 14 h-DC respect to the other time points, maybe due to synergic effect of viral pulse and cytokines used for the culture. In group B, cells significantly modulated FCAR, IL1B, IL12B, SELL and STAT1 (Fig. 3B). Of notice, expression of IL12B was augmented at the end of protocol, suggesting the ability of these DC to produce IL-12, a key cytokine in the context of immunologic synapsis. The expression of transcription factors STAT1, IRF1 and NF-KB has been reported to contribute to the altered susceptibility to HIV infection [14], for this reason we considered and plotted the expression profile of those genes during DC manipulation (Fig. 3C and D), even if FC or p-value were out of our selection criteria. It is interest to notice that whether NFKB was weakly up-regulated in both groups, STAT1, and IRF1 were down-regulated only in group A. As IL12 gene is a target of IRF1, it is not surprising that only B group cells showed up-regulation of IL12 (Fig. 3B). Considering above-mentioned data about IL1B differential gene expression (Fig. 3), and the key role of this cytokine in DC biology as well as our previously reported data about the constitutive expression of inflammasome genes in DC from HIV+ individuals [15], we evaluated the expression of inflammasome’ genes NLRP3, CASP1 and IL18 in monocyte-to-DC differentiation with a gene specific probe assay. No significant difference in NLRP3 or CASP1 modulation was observed in DC compared to monocytes in the two groups (Fig. 4), compatible with IL1B expression data (Fig. 3) and in accordance with our previously published results [15]. Unexpectedly IL18 was up-regulated in DC from group A donors, at 14 as well as at 48 h (2.38 and 1.94-fold, respectively), but not in group B cells (−0.29 and 0.42-fold, respectively). The difference between IL18 expression in 48 h-DC of groups A and B resulted statistically significant (p < 0.05). Considering the difference of anti-HIV response genes expression between the two groups of patients, we wondered whether, before starting differentiation protocol, ex vivo peripheral blood monocytes just show a different expression profile. For this purpose, differential gene expression was evaluated compared group B versus group A monocytes, and only one gene, CCR5, resulted significantly up-regulated (16-fold, p = 1.99exp-4). This data suggests that monocytes belonging to group B could be more chronically activated [16] and this condition could affect also the chronic activation state of respective monocyte-derived dendritic cells, as we observed in B group DC (Table 3).