The human CD5L/AIM-CD36 axis: A novel autophagy inducer in macrophages that modulates inflammatory responses

<div><p>CD5L (CD5 molecule-like) is a secreted glycoprotein that participates in host response to bacterial infection. CD5L influences the monocyte inflammatory response to the bacterial surface molecules lipopolysaccharide (LPS) and lipoteichoic acid (LTA) by inhibiting TNF secretion. Here we studied the intracellular events that lead to macrophage TNF inhibition by human CD5L. To accomplish this goal, we performed functional analyses with human monocytic THP1 macrophages, as well as with peripheral blood monocytes. Inhibition of phosphatidylinositol 3-kinase (PtdIns3K) reversed the inhibitory effect of CD5L on TNF secretion. Among the various PtdIns3K isoforms, our results indicated that CD5L activates PtdIns3K (whose catalytic subunit is termed PIK3C3), a key modulator involved in autophagy. Further analysis revealed a concomitant enhancement of autophagy markers such as cellular LC3-II content, increased LC3 puncta, as well as LC3-LysoTracker Red colocalization. Moreover, electron microscopy showed an increased presence of cytosolic autophagosomes in THP1 macrophages overexpressing CD5L. Besides preventing TNF secretion, CD5L also inhibited IL1B and enhanced IL10 secretion. This macrophage anti-inflammatory pattern of CD5L was reverted upon silencing of autophagy protein ATG7 by siRNA transfection. Additional siRNA experiments in THP1 macrophages indicated that the induction of autophagy mechanisms by CD5L was achieved through cell-surface scavenger receptor CD36, a multiligand receptor expressed in a wide variety of cell types. Our data represent the first evidence that CD36 is involved in autophagy and point to a significant contribution of the CD5L-CD36 axis to the induction of macrophage autophagy.</p></div

A multi-antigenic MVA vaccine increases efficacy of combination chemotherapy against <i>Mycobacterium tuberculosis</i>

<div><p>Despite the existence of the prophylactic Bacille Calmette-Guérin (BCG) vaccine, infection by <i>Mycobacterium tuberculosis</i> (Mtb) remains a major public health issue causing up to 1.8 million annual deaths worldwide. Increasing prevalence of Mtb strains resistant to antibiotics represents an urgent threat for global health that has prompted a search for alternative treatment regimens not subject to development of resistance. Immunotherapy constitutes a promising approach to improving current antibiotic treatments through engagement of the host’s immune system. We designed a multi-antigenic and multiphasic vaccine, based on the Modified Vaccinia Ankara (MVA) virus, denoted MVATG18598, which expresses ten antigens classically described as representative of each of different phases of Mtb infection. <i>In vitro</i> analysis coupled with multiple-passage evaluation demonstrated that this vaccine is genetically stable, <i>i</i>.<i>e</i>. fit for manufacturing. Using different mouse strains, we show that MVATG18598 vaccination results in both Th1-associated T-cell responses and cytolytic activity, targeting all 10 vaccine-expressed Mtb antigens. In chronic post-exposure mouse models, MVATG18598 vaccination in combination with an antibiotic regimen decreases the bacterial burden in the lungs of infected mice, compared with chemotherapy alone, and is associated with long-lasting antigen-specific Th1-type T cell and antibody responses. In one model, co-treatment with MVATG18598 prevented relapse of the disease after treatment completion, an important clinical goal. Overall, results demonstrate the capacity of the therapeutic MVATG18598 vaccine to improve efficacy of chemotherapy against TB. These data support further development of this novel immunotherapeutic in the treatment of Mtb infections.</p></div

Statistical analyses of bacterial load of pooled post-exposure experiments performed in both BALB/c and C57BL/6 mouse models.

<p>Groups of mice co-treated with antibiotics (ATB) and MVATG18598 or MVATGN33.1 given thrice subcutaneously during antibiotic therapy were analyzed.</p

MVATG18598 immunization triggers Mtb antigen-specific cells producing IFNγ and induces cells with antigen-specific cytolytic activity.

<p>IFNγ ELISpot responses specific to Mtb antigens following injection of MVATG18598 in <b>(A)</b> BALB/c and <b>(B)</b> C57BL/6 mice. Mice were immunized once with MVATGN33.1 (grey) or MVATG18598 (orange). Results are shown as the number of IFNγ-producing cells per 10⁶ splenocytes (spots/10⁶ cells) following stimulation or not (Medium) with either an irrelevant peptide (Irrelevant) or individual pools of peptides (P) of Mtb antigens expressed by MVATG18598. Each symbol represents individual mice and black lines represent median values of each group (5 or 7 mice/group). The dotted line represents the cut-off value, above which a response was considered as positive (91 and 102 spots/10⁶ cells in (A) and (B), respectively). For each mouse strain, results are representative of independent experiments. <b>(C)</b> CTL killing response to the antigen peptide-pulsed targets in mice immunized with MVATG18598. CB6F1 mice were immunized with either MVATGN33.1 or MVATG18598. Results are expressed as mean ± S.D. of the percentage (%) of specific killing of target cells present in MVATG18598-vaccinated mice minus to those in the MVATGN33.1-vaccinated mice. For each antigen peptide pool (P), statistically significant lysis is indicated by * using a permutation resampling test. <i>p</i> value<0.05.</p

Evaluation of immunization routes and positioning of MVATG18598 in the post-exposure BALB/c mouse model.

<p><b>(A)</b> Scheme of immunotherapy experiments. BALB/c mice were infected with a low dose aerosol of <i>M</i>. <i>tuberculosis</i> H37Rv. Four weeks later, mice were treated with an antibiotic regimen (RHZ) for 11 weeks. A subset of antibiotic-treated mice was vaccinated, through subcutaneous (SC) or intranasal (IN) routes, three times with a 3-week interval, with either MVATGN33.1 or MVATG18598 during (Weeks 7, 10 and 13) or after (Weeks 17, 20 and 23) chemotherapy. Twelve weeks after the end of the antibiotic regimen (Week 27), the number of viable bacteria in the lungs of animals was determined. <b>(B)</b> <i>M</i>. <i>tuberculosis</i> CFU counts after the completion of the antibiotic regimen in mice vaccinated or not (black) with either MVATGN33.1 (grey) or MVATG18598 (orange). Each symbol represents CFU value of individual mice. Dotted line indicates the lower limit of detection. Results are expressed as mean (black line) log₁₀ CFU ± S.E.M. Statistical analysis was performed using a Mann-Whitney test. *, <i>p</i><0.05. <b>(C)</b> Titers of antibodies specific to MVATG18598-expressed antigens at the relapse evaluation. Each symbol represents antibody titer (log₁₀) of individual mice vaccinated or not (black) with either MVATGN33.1 (grey) or MVATG18598 (orange). Black line represents median value for each group. Statistical analysis was performed using a Mann-Whitney test. §, <i>p</i><0.05 and §§, <i>p</i><0.01 for comparison with RHZ group. *, <i>p</i><0.05 and **, <i>p</i><0.01 for comparison between MVATGN33.1- and MVATG18598-vaccinated groups.</p

Schematic representation of antigenic expression cassettes in MVATG18598 and <i>in vitro</i> detection of antigen expression.

<p><b>(A)</b> MVATG18598 contains the fusion Rv2626/T2a/Ag85B under the control of pB2R promoter, the fusion CFP10/ESAT6 under the control of pH5R promoter, the fusion TB10.4/Rv0287 under the control of pSE/L promoter, the fusion RpfB-RpfD under the control of p7.5K promoter and the fusion Rv3407/E2a/Rv1813 under the control of pA35R promoter. <b>(B)</b> CEF cells were infected or not (Mock) with MVATG18598 or MVATGN33.1 and cell extracts analyzed by Western blot. Fusion Rv2626/T2a/Ag85B was detected using a mouse monoclonal anti-Rv2626 antibody (expected molecular weight of cleaved product: 17.4 kDa) and a rabbit polyclonal anti-Ag85B antibody (expected molecular weight of cleaved product: 31.3 kDa). CFP10/ESAT6 fusion (expected molecular weight: 21.8 kDa) was detected using a mouse monoclonal anti-ESAT6 antibody. TB10.4/Rv0287 fusion (expected molecular weight: 21.2 kDa) was detected using an anti-TB10.4 rabbit polyclonal antibody. Fusion RpfB-RpfD (expected molecular weight: 37.0 kDa) was detected using a rabbit polyclonal anti-RpfB antibody. Fusion Rv3407/E2a/Rv1813 was detected using a rabbit polyclonal anti-Rv3407 antibody (expected molecular weight of cleaved product: 13.2 kDa) and a rabbit polyclonal anti-Rv1813 antibody (expected molecular weight of cleaved product: 12.1 kDa). ND; not done.</p

Percentage of relapsing mice and bacterial burden in mice vaccinated SC with increasing vaccination number of MVATG18598 during antibiotic regimen.

<p>Percentage of relapsing mice and bacterial burden in mice vaccinated SC with increasing vaccination number of MVATG18598 during antibiotic regimen.</p

Percentage of relapsing mice and bacterial burden in mice vaccinated SC or IN with MVATG18598 or empty vector, MVATGN33.1, during or after antibiotic regimen.

<p>Percentage of relapsing mice and bacterial burden in mice vaccinated SC or IN with MVATG18598 or empty vector, MVATGN33.1, during or after antibiotic regimen.</p

Percentage of relapsing mice and bacterial burden in mice vaccinated SC with MVATG18598 during antibiotic regimen.

<p>Percentage of relapsing mice and bacterial burden in mice vaccinated SC with MVATG18598 during antibiotic regimen.</p

Efficacy of MVATG18598 in the post-exposure C57BL/6 mouse model.

<p><b>(A)</b> Scheme of immunotherapy experiments. C57BL/6 mice were infected and treated with an antibiotic regimen (P, rifapentine and H, isoniazid) as described in Material and Methods section. Two subsets of antibiotic-treated mice received 3 injections of either MVATGN33.1 or MVATG18598 through SC route during chemotherapy (Weeks 9, 12 and 15). Seven weeks after end of antibiotic regimen (Week 23), number of viable bacteria in the lungs of animals was determined and Ag-specific immune responses were assayed. <b>(B)</b> <i>M</i>. <i>tuberculosis</i> CFU counts in the lungs after the completion of the antibiotic regimen alone (PH, black) or in combination with MVATGN33.1 (grey) or MVATG18598 (orange). Each symbol represents the CFU value of an individual mouse. Results are expressed as mean (black line) log₁₀ CFU ± S.E.M. Statistical analysis was performed using a Mann-Whitney test. *, <i>p</i><0.05. <b>(C)</b> IFNγ responses following stimulation of splenocytes from MVATGN33.1- or MVATG18598-vaccinated C57BL/6 mice with Mtb antigen-specific peptides. Cells were stimulated or not with pool of peptides specific to the Ag85B (Pool 3), RpfB-RpfD (Pool 3) or ESAT6 for IFNγ ELISpot assay as described in Material and Methods section. Results are shown as the number of IFNγ-producing cells per 10⁶ splenocytes (spots/10⁶ cells) following stimulation or not (Unstimulated) with pools of peptides. Each experimental group is composed of 8 mice resulting from two cumulated runs with 4 mice per run randomly selected at the end of the <i>in vivo</i> experiment part. Each symbol represents an individual mouse and black lines represent median values of each group. Statistical analysis was performed using a Mann-Whitney test: *, <i>p</i><0.05; **, <i>p</i><0.01 and ***, <i>p</i><0.001. <b>(D)</b> Titers of antibodies specific to MVATG18598-expressed antigens at the relapse evaluation. Each symbol represents antibody titer (log₁₀) of individual mice vaccinated or not (black) with either MVATGN33.1 (grey) or MVATG18598 (orange). Black line represents median value for each group. Statistical analysis was performed using a Mann-Whitney test. §§, <i>p</i><0.01 and §§§, <i>p</i><0.001 for comparison with RHZ group. ***, <i>p</i><0.001 for comparison between MVATGN33.1- and MVATG18598-vaccinated groups.</p