Binding of JunB and NF-κB/p65 to the promoters of TNFα, IL-6 and IL-12p40 genes in LPS-stimulated BMDCs.

<p>(A) <i>Binding sites for AP-1 and NF-κB in the promoter regions of TNFα, IL-6 and IL-12p40</i>. The grey bars with the inverted arrows (enh) indicate the enhancer-containing fragment which is amplified in ChIP experiments to visualize JunB and NF-κB binding. The black bars with the inverted arrows (cont) located downstream of the various genes indicate the amplified negative control fragments used in the ChIP experiments to exclude non-specific JunB and NF-κB binding. (B, C and D) <i>Binding of JunB to the promoter regions of the TNFα, IL-6 and IL-12p40 genes</i>. BMDCs were LPS-stimulated for various periods of time and ChIP experiments were carried out for assessing the presence of JunB in the cytokine gene promoter regions. PI corresponds to negative control immunoprecipitations with preimmune sera. Non-specific binding of JunB was excluded by qPCR analysis of a DNA fragment devoid of any AP-1 site and located downstream of each gene (not shown). (E, F and G) <i>Binding of NF-κB/p65 to promoter regions of the TNFα, IL-6 and IL-12p40 genes</i>. The experiments were carried as in B, C and D, except that an anti-NF-κB/p65 antiserum was used instead of the anti-JunB one. JunB and NF-κB bindings in the enhancer region are presented in arbitrary units as well as the parallel negative control ChIPs carried out with a preimmune serum (PI). Calculations were made with respect to the amplification of the “cont” negative control fragment for each gene.</p

Characterization of <i>E. coli</i> LPS-induced BMDC maturation.

<p>(A) <i>Purity of BMDCs</i>. Bone marrow cells were cultured in the presence of GM-CSF and IL-4 for 7 days and analyzed by flow cytometry for the presence of CD11c and MHC II. (B) <i>Induction of CD40 and CD80 by LPS</i>. BMDCs were LPS-stimulated for 24 hours and analyzed for the induction of CD40 and CD80 by flow cytometry. Error bars correspond to standard deviation from 5 independent experiments. (C) <i>Cytokine induction</i>. BMDCs were LPS-stimulated for the indicated periods of time and cytokines were assayed from culture supernatant by ELISA. The presented data correspond to a representative experiment.</p

Expression of Jun proteins and <i>jun</i> mRNAs in LPS-stimulated BMDCs.

<p>(A) <i>Expression of the Jun proteins</i>. BMDCs were stimulated by LPS and c-Jun, JunB and JunD levels were assayed by immunoblotting. A representative experiment out of 5 is shown. GAPDH was used as an invariant electrophoresis loading control. JunD classically appeared as 3 bands whose exact molecular natures are still not elucidated. (B) <i>Expression of jun RNAs.</i> BMDCs were stimulated by LPS and <i>c-jun</i>, <i>junb</i> and <i>jund</i> mRNA levels were assayed by qRT-PCR. As inductions of <i>junb</i> and <i>c-jun</i> mRNAs peaked at different times ranging from. 5 to 1.5 hour post-stimulation, depending on the BMDC preparation, no error bar is presented. Instead, 1 representative experiment out of 5 is presented.</p

Direct transcriptional regulation of <i>junb</i> by NF-κB in LPS-stimulated BMDCs.

<p>(A) <i>Structure of the</i> junb <i>gene</i>. <i>junb</i> has no intron and is approximately 1900 bp long. The 4 kB sites are located within a 200 bp domain located approximately 200 bp downstream of the <i>junb</i> polyadenylation signal. The grey bar with the inverted arrows (enh) indicates the enhancer-containing fragment which is amplified in ChIP experiments to visualize NF-κB binding. The black bar with the inverted arrows (cont) located downstream the NF-kB enhancer region indicates the amplified negative control fragment used in the ChIP experiments to exclude non-specific NF-κB binding. The numbers indicate nucleotide position with respect to the transcription initiation site (+1). (B) <i>NF-κB binding in LPS-stimulated BMDCs</i>. BMDCs were stimulated for various periods of time with LPS before ChIP analysis. NF-κB binding in the enhancer region is presented in arbitrary units as well as the parallel negative control ChIPs carried out with a preimmune serum (PI). Calculations were made with respect to the amplification of the “cont” negative control fragment. The presented data are representative of 3 independent experiments (see text). (C) <i>Inhibition of NF-kB binding at the junb enhancer in the presence of BAY11-7085</i>. The same experiment as in B was conducted except that BAY11-7085 or DMSO was added together with LPS to BMDCs and incubation allowed to proceed for 1 hour.</p

Involvement of the NF-κB pathway in JunB induction in BMDCs.

<p>(A) <i>Inhibition of p65/RelA nuclear translocation by BAY11-7085</i>. BMDCs were left unstimulated or were stimulated by LPS in the presence of either BAY 11-7085 or solvent (DMSO) for 1 hour. After cell fixation, nuclei were stained with Hoescht 33342 and p65/RelA was detected by indirect immunofluorescence. (B) <i>Inhibition of JunB induction by BAY11-7085</i>. BMDCs were stimulated by LPS in the presence of either BAY 11-7085 or solvent (DMSO) for various periods of time. The levels of IκBα and JunB were assayed by immunoblotting, taking GAPDH as an invariant electrophoresis loading control. The experiments were reproduced 3 times.</p

Oligonucleotide sequences for qRT-PCR (A) and ChIP (B) experiments.

<p>Oligonucleotide sequences for qRT-PCR (A) and ChIP (B) experiments.</p

Image_1_Fc receptors are key discriminatory markers of granulocytes subsets in people living with HIV-1.pdf

IntroductionGranulocytes are innate immune cells that play a key role in pathogen elimination. Recent studies revealed the diversity of granulocytes in terms of phenotype and function. In particular, a subset of granulocytes identified as low-density granulocytes (LDG) has been described in physiological conditions and with increased frequencies in several pathological contexts. However, the properties of LDG are still controversial as they vary according to the pathophysiological environment. Here we investigated the heterogeneity of granulocyte populations and the potential differences in phenotype and immunomodulatory capacity between LDG and normal density granulocytes (NDG) in people living with HIV-1 (PLWH).MethodsTo this end, we developed an optimized method to purify LDG and NDG from a single blood sample, and performed in-depth, comparative phenotypic characterization of both granulocyte subtypes. We also assessed the impact of purification steps on the expression of cell surface markers on LDG by immunophenotyping them at different stages of isolation.ResultsWe identified 9 cell surface markers (CD16, CD32, CD89, CD62L, CD177, CD31, CD10, CXCR4 and CD172α) differentially expressed between LDG and NDG. Noteworthy, markers that distinguish the two subsets include receptors for the Fc part of IgG (CD16, CD32) and IgA (CD89). Importantly, we also highlighted that the purification procedure affects the expression of several cell surface markers (i.e.CD63, CD66b, …) which must be taken into account when characterizing LDG. Our work sheds new light on the properties of LDG in PLWH and provides an extensive characterization of this granulocyte subset in which Fc receptors are key discriminatory markers.</p

Table_2_Fc receptors are key discriminatory markers of granulocytes subsets in people living with HIV-1.docx

IntroductionGranulocytes are innate immune cells that play a key role in pathogen elimination. Recent studies revealed the diversity of granulocytes in terms of phenotype and function. In particular, a subset of granulocytes identified as low-density granulocytes (LDG) has been described in physiological conditions and with increased frequencies in several pathological contexts. However, the properties of LDG are still controversial as they vary according to the pathophysiological environment. Here we investigated the heterogeneity of granulocyte populations and the potential differences in phenotype and immunomodulatory capacity between LDG and normal density granulocytes (NDG) in people living with HIV-1 (PLWH).MethodsTo this end, we developed an optimized method to purify LDG and NDG from a single blood sample, and performed in-depth, comparative phenotypic characterization of both granulocyte subtypes. We also assessed the impact of purification steps on the expression of cell surface markers on LDG by immunophenotyping them at different stages of isolation.ResultsWe identified 9 cell surface markers (CD16, CD32, CD89, CD62L, CD177, CD31, CD10, CXCR4 and CD172α) differentially expressed between LDG and NDG. Noteworthy, markers that distinguish the two subsets include receptors for the Fc part of IgG (CD16, CD32) and IgA (CD89). Importantly, we also highlighted that the purification procedure affects the expression of several cell surface markers (i.e.CD63, CD66b, …) which must be taken into account when characterizing LDG. Our work sheds new light on the properties of LDG in PLWH and provides an extensive characterization of this granulocyte subset in which Fc receptors are key discriminatory markers.</p

Image_2_Fc receptors are key discriminatory markers of granulocytes subsets in people living with HIV-1.pdf

IntroductionGranulocytes are innate immune cells that play a key role in pathogen elimination. Recent studies revealed the diversity of granulocytes in terms of phenotype and function. In particular, a subset of granulocytes identified as low-density granulocytes (LDG) has been described in physiological conditions and with increased frequencies in several pathological contexts. However, the properties of LDG are still controversial as they vary according to the pathophysiological environment. Here we investigated the heterogeneity of granulocyte populations and the potential differences in phenotype and immunomodulatory capacity between LDG and normal density granulocytes (NDG) in people living with HIV-1 (PLWH).MethodsTo this end, we developed an optimized method to purify LDG and NDG from a single blood sample, and performed in-depth, comparative phenotypic characterization of both granulocyte subtypes. We also assessed the impact of purification steps on the expression of cell surface markers on LDG by immunophenotyping them at different stages of isolation.ResultsWe identified 9 cell surface markers (CD16, CD32, CD89, CD62L, CD177, CD31, CD10, CXCR4 and CD172α) differentially expressed between LDG and NDG. Noteworthy, markers that distinguish the two subsets include receptors for the Fc part of IgG (CD16, CD32) and IgA (CD89). Importantly, we also highlighted that the purification procedure affects the expression of several cell surface markers (i.e.CD63, CD66b, …) which must be taken into account when characterizing LDG. Our work sheds new light on the properties of LDG in PLWH and provides an extensive characterization of this granulocyte subset in which Fc receptors are key discriminatory markers.</p

Table_1_Fc receptors are key discriminatory markers of granulocytes subsets in people living with HIV-1.docx

IntroductionGranulocytes are innate immune cells that play a key role in pathogen elimination. Recent studies revealed the diversity of granulocytes in terms of phenotype and function. In particular, a subset of granulocytes identified as low-density granulocytes (LDG) has been described in physiological conditions and with increased frequencies in several pathological contexts. However, the properties of LDG are still controversial as they vary according to the pathophysiological environment. Here we investigated the heterogeneity of granulocyte populations and the potential differences in phenotype and immunomodulatory capacity between LDG and normal density granulocytes (NDG) in people living with HIV-1 (PLWH).MethodsTo this end, we developed an optimized method to purify LDG and NDG from a single blood sample, and performed in-depth, comparative phenotypic characterization of both granulocyte subtypes. We also assessed the impact of purification steps on the expression of cell surface markers on LDG by immunophenotyping them at different stages of isolation.ResultsWe identified 9 cell surface markers (CD16, CD32, CD89, CD62L, CD177, CD31, CD10, CXCR4 and CD172α) differentially expressed between LDG and NDG. Noteworthy, markers that distinguish the two subsets include receptors for the Fc part of IgG (CD16, CD32) and IgA (CD89). Importantly, we also highlighted that the purification procedure affects the expression of several cell surface markers (i.e.CD63, CD66b, …) which must be taken into account when characterizing LDG. Our work sheds new light on the properties of LDG in PLWH and provides an extensive characterization of this granulocyte subset in which Fc receptors are key discriminatory markers.</p