**3. Results**

#### *3.1. Di*ff*erent Replicative Kinetics of CXCR4- and CCR5-Dependent HIV Strains in MDM*

The first step of this study was to evaluate the efficiency of CCR5-, CXR4- tropic HIV-1 strains to replicate in MDM by measuring p24 production (Figure 1). In particular, p24 production was similar up to day 7 post infection for both CCR5- and CXR4-tropic strains. After 7 days post infection, p24 production of the CCR5-tropic 81A virus underwent a sharp increase up to day 10 and then tended to remain stable up to day 14. Conversely, p24 production of the CXCR4-tropic NL4.3 sharply decreased, becoming almost undetectable starting from day 10 (Figure 1). As a control, pre-treatment with AMD3100 completely abrogated the replication of the CXCR4- tropic NL4.3 in MDM.

**Figure 1.** Kinetics of the HIV-1 replication profile in human primary monocyte-derived macrophages (MDM) infected by CXCR4-tropic NL4.3 and or CCR5-tropic 81.A virus. The figure reports p24 production starting from day 7 post-infection. MDM from a healthy donor were infected with a standard dose of p24 gag (3000 pg/mL) of NL4.3 or 81.A. p24 production was measured daily in culture supernatants by a commercially available ELISA (Abbott labs, Pomezia, Italy). Pre-treatment with AMD3100 was performed 1 h before incubation with NL4.3. All tests were performed in triplicate.

#### *3.2. E*ff*ect of CXCR4-Tropic NL4.3 and*/*or CCRR5-Tropic 81.A Dependent HIV Infection Upon DNA Fragmentation*

The second step of this study was to investigate the impact of CCR5-tropic 81.A and CXCR4-tropic NL4.3 on inducing apoptosis in MDM by cytofluorometry. In MDM infected with the CXCR4-tropic NL4.3, the percentage of cells in apoptosis progressively increased from day 4 up to day 10 post infection (43.5%) (time point at which the p24 production of the CXCR4-tropic NL4.3 becomes undetectable) and tended to remain stable at around 40% 13 days post infection (Figure 2). As a control, pre-treatment with AMD3100 abrogated the capability of CXCR4-tropic NL4.3 to induce MDM apoptosis (Figure 2). Conversely, very low levels of apoptosis were observed in MDM infected with the CCR5-tropic 81A. Overall findings sugges<sup>t</sup> that the decrease in the replication of the CXCR4-tropic NL4.3 in MDM may be linked to the capability of the viral strain to induce apoptosis of MDM. The capability of CXCR4-tropic strains to favor MDM apoptosis was confirmed in the presence of another laboratory adapted CXCR4-tropic IIIB and in the presence of different CXCR4-tropic clinical isolates. Interestingly, the CCR5/CXCR4-tropic clinical isolate #6 conserved the capability to induce MDM apoptosis despite dual tropism (Figure 3).

**Figure 2.** Effects of CXCR4 and CCR5 usage on DNA fragmentation (measure of apoptosis) in MDM. MDM from a healthy donor were infected with 81A (CCR5-tropic strain) or NL4-3 (CXCR4-tropic strain). Percentage of cells undergoing DNA fragmentation was checked: (**A**) 4 days after infection (**B**) 6 days after infection; (**C**) 10 days after infection; (**D**) 13 days after infection. Pretreatment with AMD3100 was performed 1 h before incubation with NL4-3. For HIV infection in MDM, a standard dose of p24 gag (3000 pg/mL) was used. The PI fluorescence was measured by Flow Cytometry in FL2-H (FACScan, Becton Dickinson System, San josè, CA) and registered on a logarithmic scale. The figure is representative of three independent experiments. Differences in NL4-3-infected macrophages are statistically significant (*P* < 0.001, Chi Square test) compared to mock-, 81A-infected and AMD3100- treated macrophages.

**Figure 3.** Measure of DNA fragmentation in Human Primary Macrophages infected by X4-tropic virus. The PI fluorescence was measured by Flow Cytometry in FL2-H and registered on a logarithmic scale. All the tests were performed in duplicate. (**i**) NL4-3 and 81A are, respectively, a CXCR4- (X4) and CCR5 (R5)-tropic HIV strains. DNA fragmentation was analyzed to the 10th day of infection (**ii**) IIIB and Ba-L are laboratory-adapted HIV strains using, respectively, X4 and R5 strains. DNA fragmentation was analyzed to the 7th day of infection. (**iii**) #17 and #6 are, respectively, a CXCR4- and CCR5-tropic HIV-1 clinical isolates and DNA fragmentation was analyzed to the 12th day of infection. For the infection, a standard dose of p24 gag (3000 pg/mL) correspondent to 400 TCID50/mL dose of Ba-L was used. \* Differences in X4 strain infected macrophages are statistically significative (*P* < 0.001, Chi Squared test) compared to mock infected and R5 strain infected macrophages (see material and methods).

#### *3.3. Impact of CXCR4-Tropic (CXCR4) and CCR5-Tropic (CCR5) Strains in p38 Activation in MDM*

To further corroborate the capability of CXCR4-tropic strains to favor MDM apoptosis, the detection of the phosphorylated form of the mitogen-activated protein p38/MAPK (phospho- p38) was evaluated. After exposing NL4.3 for 30 min (Figure 4 Lane 9), a 2-fold increase in the detection of the phospho- p38 MAPK was observed compared to MDM exposed to p81A. Thus, NL4-3 induced the activation of the p38/MAPK since early phases of MDM infection (while no effect was observed for 81A) [62]. Similarly, an increase in the detection of the phosphorylated form of p38 was also observed after exposing the CXCR4-tropic IIIB to MDM for 30 min (Figure 5A). Conversely, the detection of the phosphorylated form of p38 was reduced when AMD3100 was used (Figure 5B).

**Figure 4.** Detection of p38 and phospho p38 by Western Blotting in cell lysates. MDM were infected with 7500 pg/mL of NL4.3 or 81.A. Blots are representative of three experiments using MDM from different donors after exposing NL4.3 and 81.A in MDM for 5, 10 and 30 min.

**Figure 5.** Detection of p38 and phospho p38 by Western Blotting. MDM were infected with 7500 pg/mL of the CXCR4-tropic IIIB and the CCR5-tropic 81.A. (**A**) Cell lysates were subjected to immunoblot analysis with antibodies specific for the total or phosphorylated forms of p38 MAPK (phospho-p38 MAPK [T180/Y182] antibody). (**B**) MDM from HIV-1 negative donor were pretreated for 30 min with (+) or without (−) AMD3100 (5μg/mL) before incubation with IIIB. Blots are representative of three experiments using MDM from different donors.

#### *3.4. Modulation of Expression of Genes Correlated with Apoptotic Pathways, in a Time-Dependent Manner, by CXCR4-Tropic Strains*

As a final step of this study, we analyzed variation in the transcriptome profile observed in MDM infected by CCR5-tropic or CXCR4-tropic strains. We found that NL4-3, but not 81A, up-regulated many genes in human MDM, including TNF2, Fas (TNFRSF6)-associated via death domain [63], caspase-7, Cytocrome C, KGF [64] and GSPT1/eRF3 [65] but down-regulates survival and cancer genes. These findings indicate that CXCR4-mediated the entry in MDM can up regulate apoptosis-related genes and simultaneously down modulate survival-related genes as Defender against cell death 1(DAD-1) and Cullin 2 (hCUL2), involved in MDM survival after HIV-1 infection. Interestingly, we also observed enhancement in gene activation of pro-inflammatory Matrix metalloproteinase 9 (MMP9 gelatinase B, 92kD gelatinase, 92kD type IV collagenase) by the CXCR4-tropic NL4.3 but not by CCR5-tropic p81.A (Figure 6 and Table S1).

**Figure 6.** Photographs of arrays representing transcriptional changes in macrophages infected by 81A and NL4-3 strains. In array (**A**) the Cy3 spots indicated by numbered squares represent the apoptosis-related genes activated by NL4-3 whereas in subarray (**B**) the Cy5 spots indicated by numbered squares represent the survival-related genes activated by 81A. The square numbers relative to the genes are also indicated in Table S1.
