1. Hedlund S, et al. (2010) Dendritic cell activation by sensing Mycobacterium tuberculosis–induced apoptotic neutrophils via DC-SIGN, Hum Immunol, doi: 10.1016/j.humimm.2010.02.022.  Mycobacterium tuberculosis (Mtb) is one of the most prevalent infectious agents, able to live intracellularly.  Only 10% of infections develop into tuberculosis.  It has been shown that Mtb adaptive immunity is mainly initiated in the lymph nodes and not in the lungs, where Mtb infections occur.  PMNs undergo apoptosis with natural aging but this is accelerated when they take up Mtb.  Apoptotic cells have been shown to be drained to the lymph nodes where immature dendritic cells (IDCs) sample them for antigens to present to T-cells if induced to mature  The goal of this study was to determine the interaction of Mtb-induced apoptotic cells (PMN Mtb) and dendritic cells. Background and Study Aims

2. Experimental approach  Induction of apoptosis  Neutrophils (PMNs) were exposed to medium or opsonized Mycobacterium tuberculosis (Mtb) at a ratio of 5:1 (Mtb:PNM) for 45 minutes at 37ºC and left to enter apoptosis for 18 hours in 1% plasma medium and 5% CO2. Apoptotic PMN were quantified by Flow Cytometry testing for phosphatidyl serine, which is only expressed once a cell undergoes apoptosis  Mac-1 expression on PMNs was assessed by flow cytometry using monoclonal antibodies.  Uptake of apoptotic PMNs by IDC was assessed using microscopic quantification.  To evaluate the effect apoptotic cells exerted on DC maturation, IDC were co-incubated for 48 hours with PMN Apo or PMN Mtb at ratio of 2:1 (PMN:DC) and analyzed for the maturation markers CD83+, CD40, CD80 and CD86 (costimulatory molecules for T-cells) as well as the adhesion molecule DC-SIGN (receptor to which Mtb binds).  To evaluate the stimulating properties of PMN Mtb in apoptotic cells vs released compound involved in the maturation of DCs, IDCs were exposed to either the supernatant or the cellular fraction of apoptotic PMNs.  Analysis of DC maturation was performed using monoclonal antibodies and the use of CellQuest software  Mature DCs cytokine release was measured using flow-based CBA detection (fluorescent antibody test for cytokines) and the presence of IL-1β, IL-1, IL-6, IL-10, IL12 and tumor necrosis factor (TNF)-α in culture supernatants was determined.

3. Main Findings  IDCs only ingest apoptotic PMNs when the PMNs are vast excess (less than 5% of apoptotic cells were phagocytised in a 2:1 ratio PMN:DC) but only PMN Mtb are able to activate maturation of IDCs  i.e. DCs can distinguish between PMN with natural apoptosis and those who were induced.  PMN Mtb induce IDC maturation almost as much as exposure to LPS does, and the mature PMN Mtb induced DCs also secrete cytokines IL1-β, IL6 and TNF-α as much as LPS induced mature DCs. (PMN Apo did neither)  Neither the bacteria or excreted signal molecules in the supernanant were able to induce DC maturation.  i.e. Cell to cell contact of PMN Mtb and DC by phagocytosis is necessary for PMN Mtb induced DC maturation  The inducing of maturation is only possible there is an interaction between DC-SIGN of the DCs and the Mac-1 integrin of the PMN

4. Mature Dendritic cells induced by sample through CD83+ expression Critique  This is a well done experiment with positive and negative controls in all tests as well as tested for multiple variables.  The article has a very good discussion in which it highlights the importance of the interaction of the innate and adaptive immune responses.

5. Discussion  In this study beads coated with antibodies for cytokines were used for flow cytometry.  All of the markers for mature DCs showed the same results as the one discussed  The receptor MAC-1 is expressed on PMNs whether apoptosis was induced by mycobacterium or not.  The cytokines are not markers for mature DCs but to check if the mature DCs are functional. Ability to activate T helper cells was also measured for the same reason.  This study focused on the reaction between PMNs and DCs as the pathway of the Mtb immune response is not fully known