Impairment of T cell development and acute inflammatory response in HIV-1 Tat transgenic mice

Immune activation and chronic inflammation are hallmark features of HIV infection causing T-cell depletion and cellular immune dysfunction in AIDS. Here, we addressed the issue whether HIV-1 Tat could affect T cell development and acute inflammatory response by generating a transgenic mouse expressing Tat in lymphoid tissue. Tat-Tg mice showed thymus atrophy and the maturation block from DN4 to DP thymic subpopulations, resulting in CD4+ and CD8+ T cells depletion in peripheral blood. In Tat-positive thymus, we observed the increased p65/NF-κB activity and deregulated expression of cytokines/chemokines and microRNA-181a-1, which are involved in T-lymphopoiesis. Upon LPS intraperitoneal injection, Tat-Tg mice developed an abnormal acute inflammatory response, which was characterized by enhanced lethality and production of inflammatory cytokines. Based on these findings, Tat-Tg mouse could represent an animal model for testing adjunctive therapies of HIV-1-associated inflammation and immune deregulation.

Scientific RepoRts | 5:13864 | DOi: 10.1038/srep13864 Tat plays a major role in inflammation and immune dysfunction in AIDS by altering the cytokine network 6,7 and causing apoptosis of CD4 + T-cells 8 and neurons 6,9 . In human monocytes and macrophages, Tat enhances the expression of pro-inflammatory cytokines, such as IL1-β , IL-6, IFN-α , IFN-γ , and TNF-α , and the immunosuppressive Th2 cytokine IL-10 [10][11][12][13] , which indeed must be strictly and timely regulated to ensure the appropriate immune control of pathogens. In the gastrointestinal tract, HIV-1 infection causes immune hyper-activation, which results in the breakdown of mucosal barrier with the release of bacterial products in the blood to sustain chronic inflammation 2 . Tat also promotes severe microbial infections in AIDS by promoting the expression of co-infecting opportunistic pathogens, including Herpes simplex virus-1 (HSV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), Human Papilloma Virus (HPV), Mycobacterium avium and Mycobacterium tubercolosis, protozoa of Trypanosomatidae family, and Candida albicans 14 .
NF-κ B transcription factors regulate the expression of genes involved in the immune response, inflammation and cell survival 15 . Inhibitors of NF-κ B (Iκ Bs) repress the NF-κ B activity by associating with NF-κ B dimers and impeding their binding to DNA 16 . Appropriate signalling causes the ubiquitination-coupled to proteasomal degradation of Iκ B repressors, releasing the NF-κ B complexes for gene expression activation 17 . The NF-κ B activity is strictly regulated to avoid the abnormal expression of NF-κ B-dependent genes. Most cytokines involved in the development of immune system and immune control of pathogens are under the transcriptional control of NF-κ B. NF-κ B deregulation leads to immunological disorders, such as autoimmunity and immunodeficiency, as a consequence of altered expression of immunity functions. As mechanism of NF-κ B deregulation in HIV-1 infection, we previously demonstrated that Tat up regulated the NF-κ B activity through its physically association with the Iκ B-α inhibitor, impeding the repressor-mediated shut-off of NF-κ B 18 . Moreover, Tat bound to the p65 subunit of NF-κ B interfering the Iκ B-a repressor binding, and increasing the p65 recruitment to the NF-κ B enhancer 18 . Further, in HIV-1-infected monocytes Tat enhanced the NF-κ B-dependent expression of genes of immune response genes, including MIP-1α , CSF3, LTA, NFKBIA and TLR2 18 , which was consistent with similar findings in the sera of HIV-1 patients 19,20 . It is worthwhile to note that Tat presents some functional similarities with the HTLV-1-encoded Tax protein, which deregulates the NF-κ B activity and affects cell proliferation, cell-cycle and apoptosis [21][22][23] .
Based on Tat-mediated deregulation of NF-κ B, we addressed the question whether Tat could affect the development of lymphoid tissues and the acute inflammatory response in mice. To this end, we generated a transgenic mouse carrying the HIV-1 Tat gene under the control of the H2-K b promoter and immunoglobulin heavy chain enhancer, which allowed to direct the Tat expression in lymphoid tissues.

Results
Impaired thymocytes differentiation in Tat-Tg mice. To analyse the effects of Tat on the immune system, we generated a transgenic mouse carrying the HIV-1 Tat 1-86 nucleotide sequence under the transcriptional control of the H2-K b promoter and immunoglobulin heavy chain enhancer (Fig. 1A). As measured by qRT-PCR of total RNA, Tat was mainly expressed in the thymus and to a lesser extent in the spleen, lymph nodes, bone marrow and lung (Fig. 1B). In the spleen, Tat expression was observed in T-cells (CD3 + CD4 + and CD3 + CD8 + ) and B-cells (B220 + ). Differently, Tat was not expressed in the heart, kidney and liver (Fig. 1B). The expression of Tat protein in the thymus and spleen was confirmed by western blotting analysis (Fig. 1C). Tat was undetected in the serum of Tat-Tg mice by ELISA ( Figure S1).
Tat-Tg mice were viable and fertile, and did not show any behaviour or gross anatomical alterations, except a significant reduction of thymus (size and weight) and total number of thymocytes ( Fig. 2A-C). No differences were observed for the spleen ( Fig. 2A-C). In Tat-Tg mice, the histological analysis showed the atrophy and altered architecture of thymus, including lack of lobular definition, cortex size reduction with faintly discernible cortical-medullary boundaries, and several cysts increasing with the animal age ( Figure S2). Differently, the spleen architecture of Tat-Tg mice was unaffected ( Figure S3).
T cell development occurs through sequential steps in the thymus, where CD4 − CD8 − double negative (DN) cells progress toward CD4 + CD8 + double positive (DP) cells, which then differentiate toward CD4 + or CD8 + single positive (SP CD4 or SP CD8, respectively) T cells. In 8 weeks old Tat-Tg mice, we observed a percentage increase of DN cells (12.6% in Tat-Tg vs 4.1% in wild type), with decrease of DP cells (65.6% in Tat-Tg vs 79.7% in wild type), without significant differences of SP CD4 and SP CD8 cells (Fig. 2D). The absolute number of DP, SP CD4 and SP CD8 subpopulations was significantly reduced in Tat-Tg mice (Fig. 2E). Due to the increase of DN subpopulation, we investigated the sequential steps of DN maturation toward DP cells by FACS analysis of CD44 and CD25 expression in gated CD3-CD4-CD8-cells, according to the classification of DN1 (CD44 + CD25 − ), DN2 (CD44 + CD25 + ), DN3 (CD44 − CD25 + ) and DN4 (CD44 − CD25 − ) cells. Tat-Tg mice showed a percentage decrease of DN1 and DN3 subpopulations, and increase of DN4 cells (Fig. 2F), with a statistically significant augmentation of DN4 absolute number (Fig. 2G). In the spleen, no statistically significant difference was observed in percentage and absolute number of SP CD4, SP CD8 and B220+ B-cells sub-populations ( Figure S4). These data indicated that Tat, even though expressed in both thymus and spleen, exclusively affected the thymus structure and thymocytes differentiation from DN4 to DP step. Deregulation of NF-κB activity and cytokines/chemokines expression in the thymus of Tat-Tg mice. Thymocytes differentiation toward mature T lymphocytes is timely regulated by cytokines and chemokines that are produced at specific maturation steps [24][25][26] . Thus, we asked the question whether Tat affected the cytokines and chemokines expression in the thymus as mechanism of altered T-lymphopoiesis. The expression profile of 90 cytokines and chemokines was analysed by qRT-PCR of total RNAs extracted from thymus of 8 weeks old mice (Supplementary Table 1). Several cytokines and chemokines were up regulated (CCL3, CCL4, IL-10, INFG and CCL7), or down regulated (CCL11, CCL19, CCL20, CCL25, CCL8, CXCL11, CXCL12, CXCL5, IL13RA1, IL1R1, IL1R2, IL8RB) in Tat-Tg mice (Fig. 3A). The enhanced expression of IL-10 and INFG, and down-regulation of CCL25, and CXCL5 were confirmed at the protein level by western blotting analysis (Fig. 3B).
Consistently with the enhanced expression of inflammatory cytokines under NF-κ B transcriptional control 18,27,28 , an increased p65 and p50 NF-κ B DNA binding activity was observed in Tat-positive thymus as compared to wild type (Fig. 3C). These results suggested that the aberrant expression of cytokines/ chemokines in the thymus of Tat-Tg mice likely affected the thymocytes differentiation.
Altered microRNAs expression profile in the thymus of Tat mice. MicroRNA (miRNAs) regulate mRNAs expression at post-transcriptional level during thymus development and involution [29][30][31] . Tat was shown to modify miRNAs expression by acting as transcriptional transactivator of miRNA genes 32 , or as suppressor of RNA silencing (SRS) through inhibition of Dicer activity 33 . Thus, we analysed the genome-wide miRNAs expression profile in the thymus of wild type and Tat-Tg mice. A significant increase of 5 miRNAs and decrease of 17 miRNAs occurred in Tat-Tg mice (Fig. 3D). In particular, the up-regulation of microRNA-709, microRNA-320 and microRNA-128a, and down-regulation of microRNA-181a-1-3p, microRNA-30b and microRNA-374 were confirmed by RT-PCR (Fig. 3E). These results indicated that Tat modified the expression of specific miRNAs in the thymus, which could be relevant for T-lymphopoiesis.
Peripheral blood lymphocytes and serum cytokines and chemokines in Tat-Tg mice. Next, we analysed the lymphocytes population in the peripheral blood of mice. By using an automated blood cell counter, a significant reduction of total lymphocytes was observed in Tat-Tg mice as compared to wild type (Fig. 4A,B). By FACS, we observed a significant decrease in percentage and absolute number of T-lymphocytes in the peripheral blood of Tat-Tg mice as compared to wild type ( Figure S5A; Fig. 4C,D). A slight percentage increase of B cells was also observed in Tat-Tg mice without difference in absolute number (Fig. 4C,D). Moreover, by analysing the CD3 + T-lymphocyte sub-populations, the absolute number of CD3 + CD4 + and CD3 + CD8 + T cells were strongly reduced in Tat-Tg mice (Fig. 4E,F). Gated CD3 + cells were CD45 + , and did not express the CD14 marker ( Figure S5B,C), thus excluding the contamination with other cell subpopulations, such as monocytes. As apoptosis caused depletion of CD4 + and CD8 + T cells in HIV-1 infection 34 , we analysed apoptosis of CD3 + CD4 + and CD3 + CD8 + T cells by FACS. As measured by Annexin V/7-AAD assay, no statistically significant differences in percentage and absolute number of apoptotic CD3 + CD4 + and CD3 + CD8 + T-lymphocytes were observed in wild type and Tat-Tg mice (Fig. 4G,H). These findings suggest that Tat, affecting the late stage of T-lymphopoiesis,     Table 2). In particular, CXCL13, CXCL10, CXCL1, CXCL9, TIMP-1 and TNF-α were up regulated, while CXCL12 was down regulated (Fig. 4I). These results demonstrated an increased level of circulating inflammatory cytokines in Tat-Tg mice as hallmark of chronic inflammation.
As chronic inflammation in HIV-1 infection determined the breakdown of mucosal barrier 2 , we addressed the question whether Tat affected the integrity of intestinal epithelium. Histological analysis of large bowel sections showed a normal glandular epithelium without intra-epithelial lymphocytic infiltrates in wild type and Tat-Tg mice ( Figure S6A). By immunohistochemical analysis, a few perivascular CD4 + T lymphocytes were detected in both animal groups in absence of a significant intra-epithelial component ( Figure S6A). We also observed a strong reactivity to anti-Occludin, which is a tight junction marker, thus indicating that the integrity of intestinal epithelium was unaffected in wild type and Tat-Tg mice ( Figure S6A). By TUNEL assay, there was no significant enterocytes apoptosis in the same large bowel sections of wild type and Tat-Tg mice ( Figure S6B). Altogether these results exclude a role of Tat in the breakdown of mucosal barrier.
Abnormal inflammatory response to LPS in Tat-Tg mice. Next, we asked the question whether the increased production of inflammatory cytokines together with mild reduction of circulating T cells could affect the acute inflammatory response in Tat-Tg mice. Thus, we challenged mice with lipopolysaccharide (LPS) endotoxin, which is component of gram-negative bacterial wall causing septic shock [35][36][37] . Wild type and Tat-Tg mice were intra-peritoneally injected with LPS (15 mg/Kg) and monitored for survival. At 24 hours post-injection, 40% of Tat-Tg mice survived as compared to 100% of wild type (Fig. 5A). In peritoneal lavage fluids of survived Tat-Tg mice, we observed a statistically significant increase of GM-CSF, IL-1α , IL-1RA, IL-6, IL-10, IL16, CXCL10, CXCL11, MIP-1α , MIP-1β , CXCL2, RANTES, TIMP-1 and TNF-α , and decrease of IL-7 as compared to wild type ( Fig. 5B; Supplementary  Table 3). Altogether these results indicated that Tat-Tg mice were more susceptible to LPS-induced death, and had a higher production of inflammatory cytokines, including IL-1α and TNF-α , which are key factors in the development of septic disease.

Discussion
Tat is an essential protein for HIV-1 replication and AIDS pathogenesis. Besides its role as transcriptional activator of HIV-1 and cellular genes, Tat affects the cellular metabolism through physical interaction with different cellular targets. Mostly relevant for the immune system, Tat counteracts the action of the Iκ B-α repressor of NF-κ B and increases the transcriptional activity of the p65 subunit of NF-κ B, thus enhancing the NF-κ B activity and the expression of inflammatory cytokines and chemokines 18 . As additional mechanism of inflammation, Tat promotes the expression of IL-6 and TNF-α gene through the binding to their 5′ untranslated leader mRNAs 13,38 .
NF-κ B activates the expression of genes involved in the development of immune system and immune response, and is timely regulated by the immune signalling 15 . Deregulation of the NF-κ B activity can alter the immune defence against pathogens, and eventually cause immunodeficiency or autoimmunity 15 . Based on the constitutive activation of NF-κ B by Tat 18 , we addressed the question whether Tat could affect T-lymphopoiesis and alter the acute inflammatory response to LPS. To this end, we generated a transgenic mouse that expressed HIV-1 Tat under the H2-K b promoter and immunoglobulin heavy chain enhancer to allow the specific expression of Tat in lymphoid tissues [39][40][41] . In Tat-Tg mice, Tat was peculiarly expressed in the thymus and to a lesser extent in the spleen, lymph nodes, bone marrow and lung. Thymus atrophy and thymocytes depletion was observed in Tat-Tg mice with 3D structural alterations of thymic architecture, including cystic formations arising at earlier time as compared to wild type ( Figure S2). Crosstalk signals deriving from developing thymocytes are relevant for appropriate 3D configuration of thymus structure. In fact, depletion of thymic precursors was associated with cystic involution of thymus 42 . In this regard, the early appearance of cystic formations in Tat-Tg mice could be a sign of thymus involution due to alteration of thymocytes compartments. Consistently, Tat-Tg mice had an impaired thymopoiesis with increased percentage of DN (CD4 − CD8 − ) cells, and decrease of DP (CD4 + CD8 + ), SP (CD4 + CD8 − ) and SP (CD4 − CD8 + ) cells. In particular, we observed an increased DN4 subpopulation of DN compartment of Tat-Tg mice, suggesting a partial maturation block from DN4 toward DP differentiation step. These findings are consistent with a defective thymopoiesis and thymus destruction occurring in HIV-1 infection as consequence of increased susceptibility of human thymocytes and thymic stromal cells to the virus 43 . As unique source of CD4 + and CD8 + T cells deriving from the sequential maturation steps of DN and DP cells, the thymus is essential for T-cell renewal, and is a major target of HIV-1 infection 44 . In adults, the importance of a functional thymus is not fully understood. However, the thymus is considered to be critical in HIV disease, in which T cells are depleted in large numbers. Alterations of thymic structure and function were previously reported in HIV-1 infection 45 ; in particular, histopathological and morphological evidence of thymic dysfunction occurred in infants with rapid onset of severe AIDS 46 47,48 , causing thymic pathological conditions. In humans, the thymic atrophy is a recurrent finding characterized by an increased number of DN thymocytes and depletion of DP and SP (CD4 + CD8 − ) thymocytes 46,49 . In HIV-1-infected children, loss of peripheral CD4 + and CD8 + T cells derives from a severe loss of DP cells 50 ; similar findings were observed in SIV-infected macaques 51,52 . Thus, in Tat-Tg mice, the thymic atrophy associated with increased DN population and decreased DP population resembles the alterations occurring in HIV-1/SIV infection, especially in paediatric AIDS, suggesting that Tat is a major viral protein involved in aberrant thymopoiesis in AIDS.

. HIV-1 and simian immunodeficiency virus (SIV) can infect thymocytes in vivo and in vitro
Tat-Tg mice did not develop tumours up to 18 months observation. Differently, other transgenic mice that ubiquitously expressed Tat under the SV40 promoter developed dermal lesions resembling Kaposi's sarcoma 53,54 , which suggests a potential oncogenic role of Tat depending on tissue-specific expression. Moreover, Tax-transgenic mice, where the HTLV-Tax gene expression was under the Lck promoter and restricted to thymocytes, developed T-cell leukemia-lymphoma 55 . In this regard, the lack of oncogenic activity of Tat in Tat-Tg mice here described remarks a different action of Tat as compared to Tax, as Tat mainly affects T-lymphopoiesis.
NF-κ B activity must be strictly regulated in the course of thymocytes differentiation with activity oscillations at specific maturation steps 15 . In DN compartment, a low NF-κ B activity is usually observed at DN1 and DN2 stages, followed by an increase at DN3 stage, and rapid decrease in the DN4 transition toward DP cells 15 . Consistently with Tat-dependent up-regulation of NF-κ B 18 , the p65/NF-κ B DNA binding activity and expression of NF-κ B-dependent MIP-1α , MIP-1β and INFγ genes were increased in the thymus of Tat-Tg mice. These findings suggest that the Tat-dependent up-regulation of NF-κ B likely interfered the DN4 differentiation toward DP cells, as the NF-κ B activity must be down regulated at this specific step of thymocytes differentiation 15 . As additional effect, Tat lowered the thymic expression of CXCL12 and CCL25, which are required for thymocytes maturation from DN3/DN4 to DP stage [24][25][26] . The mechanism of CXCL12 and CCL25 down-regulation could likely involve the Tat interaction with cellular functions that are involved in the CXCL12/CCL25 gene expression control.
MiRNAs regulate the gene expression at post-transcriptional level during immune system development 30,31 . Consistently with previous reports 56-58 , we found a number of differentially expressed miRNAs in the thymus of Tat-Tg mice as compared to wild type. Among others, microRNA-181a-1-3p was significantly down regulated in Tat-Tg mice. Over-expression of the precursor mir-181a-1 in DN thymic progenitors was previously shown to increase the number of DP cells 59 . In this regard, the evidence of microRNA-181a-1-3p down-regulation associated with DP cells reduction in the thymus of Tat-Tg mice suggests that the partial maturation block from DN4 to DP step could be consequence of Tat repression of mir-181a-1-3p expression. In peripheral blood, CD4 + and CD8 + T lymphocytes were significantly reduced in Tat-Tg mice. These findings could be due to the low production of mature SP CD4 + and SP CD8 + cells in the thymus of Tat-Tg mice, and are consistent with the depletion of peripheral CD4 + and CD8 + T cells in HIV-1-infected children 50 . Apoptosis caused the depletion of CD4 + and CD8 + T cells in HIV-1 infection 34 . In Tat-Tg mice, any increase in CD4 + and CD8 + T cells apoptosis was observed in peripheral blood, thus supporting the hypothesis that Tat caused a depletion of circulating T cells by mainly deregulating T-lymphopoiesis. Tat-Tg mice also showed an altered expression profile of serum cytokines and chemokines, which resembled the one reported in progressive AIDS 19,60 . In particular, we observed the increase of IL-1α IL-1Rα , TNF-α , IL-6 and CXCL10, which arise soon after HIV-1 detection and acute infection 19,60 , and the decrease of IL-10, which is peculiar of late stage of HIV-1 infection 19,60 .
Immune dysfunction and immunosuppression are hallmark features of septic syndrome and a major cause of mortality. In particular, HIV-1 patients show a higher susceptibility to septic shock as consequence of severe course of opportunistic diseases, immunodeficiency and persistent inflammation 14 . In this regard, Tat-Tg mice were more susceptible to the systemic inflammatory response syndrome caused by the intra-peritoneal injection of LPS, based on the evidence of increased lethality and expression levels of TNF-α , IL-1α , IL-6 and CXCL10 in the peritoneal lavages of surviving animals. These findings were consistent with the typical extreme "cytokines storm" characterized by chronically elevated or excessive production of inflammatory cytokines, including TNF-α , IL-1α , IL-6 and CXCL10 10 . Chronic inflammation in HIV-1 infection is an emerging cause of non-AIDS comorbidities, such as cardiovascular and cerebrovascular disease, diabetes, chronic kidney disease, osteoporosis and cancer, in patients treated with antiretroviral therapy 61 . In this perspective, the Tat transgenic mouse could represent an animal model of AIDS-related chronic inflammation and immune dysfunction, in order to test novel therapeutic strategies to counteract the Tat-dependent immune deregulation.

Methods
Plasmids. The plasmid pHSE-Tat was generated by cloning the HIV-1 Tat (1-86) cDNA of HTLVIIIB strain in the BamHI/SalI restriction sites of pHSE under the H-2K b promoter and the immunoglobulin heavy chain enhancer, which direct the expression of Tat in lymphoid tissues 39-41 . Generation of Tat transgenic mouse. The Tat transgenic mouse was generated according to established procedures [62][63][64] . Briefly, the pHSE-Tat plasmid was linearized by cutting the XhoI restriction site, and microinjected (200 ng) into the pro-nuclei of fertilized eggs from super-ovulated B6D2F1/J female mice (Charles River Laboratories, Italy). B6D2F1/J mice are a hybrid strain generated by crossing C57BL/6 females with DBA/2 males. Genomic DNA extracted from offspring tails was analysed by dot blot hybridization with the 32 P-labelled Tat cDNA for the presence of Tat gene. A founder mouse was mated with normal B6D2F1/J mice, and offspring was backcrossed for 8 generations. Heterozygous Tat transgenic mice were inter-crossed to obtain homozygous Tat mice. Animals were maintained in a dedicated pathogen-free animal facility. Mice experiments were performed with the approval of the ethical committee, as detailed in the Ethical Statement section. For genotyping, DNA was extracted from mice tails by using Extract-N-Amp Tissue PCR kit (Sigma-Aldrich). The presence of Tat gene in the genomic DNA was verified by PCR using the following primers: 5′ Tat: 5′ -TTACTCGACAGAGGAGAGCAAGAA-3′ and 3′ Tat Real-Time PCR. Total RNA was extracted from homogenized tissues with the TRIzol reagent (Invitrogen) and treated with DNase I AmpliGrade (Life Technologies, Italy), as previously described [65][66][67] . RNA aliquots (200 ng) were reverse transcribed using Random Examers (Roche Diagnostic GmbH, Mannheim, Germany) and Superscript III Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol. Real-time PCR was performed with the iQ Green Super mix (Bio-Rad Laboratories, Hercules, CA, USA) and carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) under the following conditions: 95°C, 1 min; (94°C, 10 s; 60°C, 30 s) × 40 cycles. The primers used to detect Tat expression were: forward 5′ -CCCTGGAAGCATCCAGGAA-3′ ; reverse 5′ -TCTTCGTCGCTGTCTCCGCT-3′ . To measure the cytokines and chemokines expression, RT-PCR was performed using the Mouse Cytokines & Chemokines RT 2 Profiler PCR Array profiles (QIAGEN Sciences, MD, USA). Reactions were carried out in triplicate, and gene expression levels were calculated relative to HPRT1 mRNA levels as endogenous control. Relative expression was calculated as 2(Ct gene under investigation − Ct HPRT1 ), as previously described [68][69][70][71][72] . MiRNAs expression was measured by Taqman assays using TaqMan Universal PCR Master Mix, No AmpErase UNG (Life Technologies), according to manufacturer's conditions. The following primers (Life Technologies) were used: miR-709 (RT1644 and TM1644), miR320 (RT536, TM536), miR-128a (RT453, TM453), miR-181-a1* (RT516, TM516), miR-30b (RT602, TM602), miR-374-5p (RT1319, TM1319). Sno-234 (RT1234, TM1234) and sno-429 (RT1240, TM1240) were used as internal normalizer.
For NF-κ B DNA binding analysis, nuclear extracts were collected and Electrophoretic Mobility Shift Assay (EMSA) was performed, as previously described 75  ELISA. Ninety-six wells Maxisorp microtiter plates (NUNC) were coated with 100 μ L/well containing 30 μ g of serum or thymic protein extracts of wild type or Tat-Tg mice, and incubated over-night at 4°C. The following day, the plate was washed and blocked in 200 μ L of blocking buffer (PBS 1 × , 5% non-fat dry milk, 0.01% tween 20) and next incubated in the same buffer in presence or absence of anti-Tat antibody (HIV-1 Tat mAb 5A5.3) (1 μ g/mL) for 2 h at 37 °C. Next, each well was washed six times with PBS 1 × containing 0.05% tween 20, and then incubated with a 1:5000 dilution of Alkalyne Phosphatase conjugated goat anti-mouse IgG for 1 h at 37 °C. After extensive washing, samples were incubated with PNPP (Para-Nitro-Phenyl-Phosphate) platelets substrate, diluted in Diethanolamine substrate solution pH 9.6, for 10 min at room temperature. Optical density (OD) was measured twice at 420 nm on a MULTISCAN EX ELISA Plate reader (Thermo Labsystems, Franklin, MA).  Cytokines and chemokines expression. Serum samples were obtained from blood samples of 8 weeks old mice, allowed to clot for 30 min at room temperature, and then centrifuged at 3000 × g for 15 min. Serum aliquots (150 μ L) were immediately assayed for cytokines and chemokines expression by using Mouse Cytokine Array Panel A (ARY006) (R&D System, Minneapolis, USA), according the manufacturer's conditions. Cytokines/chemokines expression levels were calculated as arbitrary units by acquiring images of developed X-ray film of cytokine array with an Image Scanner (EPSON), and measuring pixel densities of the spots with ImageJ software (ImageJ, USA). LPS challange. B6D2F1 wild type mice (4 females and 4 males) and Tat-Tg mice (5 females and 4 males) were intra-peritoneally injected with LPS (E. coli, serotype 0127:B8, Sigma) (15mg/Kg of body weight). Twenty-four hours post-injection survival rates were evaluated and surviving mice underwent to peritoneal lavages performed with 2 ml of heparinized (10 U/ml) Dulbecco's Modified Eagle Medium (DMEM). Peritoneal fluids were centrifuged at 2500 × g for 10 min, and the expression of cytokines and chemokines was measured in collected supernatants using Mouse Cytokine Array Panel A (R&D System, ARY006), according the manufacturer's protocol.

MiRNA microarray. Total
Histology, Immunohistochemistry and Fluorescence Microscopy. Thymus, spleen or large bowel of mice were wholly removed at necropsy, fixed in buffered formalin (10%) dehydrated and paraffin embedded. Thick sections (5 μ m) of each paraffin block were stained by hematoxylin and eosin or used for immunohistochemistry or fluorescent microscopy. For immunohistochemical analysis, slides were deparaffinized, rehydrated and immersed in 10 mM citric acid, pH 6, in a microwave oven (VWR International PBI Srl, Milan, Italy) to exclude epitope masking owing to fixation. Sections were immunostained with primary antibodies against CD4 and Occludin and detected with an indirect immunoperoxidase technique employing UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen (Thermo Fisher Scientific Inc., Waltham, MA, USA), according to the manufacturer's protocol. As negative controls, sections were incubated with PBS solution (Bio-Optica Milano SpA). Nuclear staining was carried out using hematoxylin. Microscopic analysis was performed with a Leica DMLB microscope (Leica Microsystems, Wetzlar, Germany). Images were taken in bright field with a digital camera (Leica DC200; Leica Microsystems) connected to the microscope. For fluorescence microscopy, slides were stained using In Situ Cell Death Detection Kit, TMR red (Roche Diagnostics), following manufacturer's instructions. Mounting was performed using Vectashield Antifade Mounting Medium with DAPI (Vector laboratories). Signals were visualized with a Leica DMLB fluorescence microscope. Images were processed as one-color images, merged and reconstructed using Adobe Photoshop software. Statistical analysis. Statistical analysis was performed by the two-tailed unpaired Student's t test.
Data were reported as mean values ± SE. Differences between the means were accepted as statistically significant at the 99% level (p < 0.01).