Inflammation and calcification are common events in chronic inflammatory diseases, such as atherosclerosis and osteoarthritis, involving infiltration of monocytes and accumulation of macrophages [1–5]. The interplay between inflammatory and pathological calcification pro- cesses is currently widely accepted, and macrophages are known key players signaling extracel- lular matrix (ECM) degradation, resident tissue cells differentiation and calcification [2, 6]. Although many aspects concerning the molecular mechanisms involved in pathological calcifi- cation remain to be elucidated, features such as chronic inflammation, increased extracellular matrix (ECM) remodeling, loss of anticalcific mechanisms leading to proliferation and differ- entiation of resident cells, and the release of calcifying extracellular vesicles (EVs) are known features contributing to the development of calcific lesions [7–11]. In atherosclerosis, early pla- que calcification associates with macrophage accumulation, and macrophage infiltration and inflammation have been shown to precede osteogenic conversion of vascular smooth muscle cells (VSMCs) and the release of EVs [12, 13]. In osteoarthritis, synovium inflammatory and destructive responses, leading to increased cartilage degradation and calcification, are largely promoted by activated synovial macrophages [14–16]. Activated macrophages at sites of tissue damage produce high levels of matrix metalloproteinases, cysteine endoproteases, cytokines, and catabolic prostaglandins which will enhance elastin and collagen degradation leading to remodeling and structural changes of the ECM, promoting calcification [12, 17, 18]. Moreover, macrophages have been shown to regulate vascular calcification through the release of osteo- genic factors capable of inducing VSMCs osteochondrogenic differentiation [2, 6]. It has been recently proposed that macrophages release calcifying EVs loaded with mineralization related factors, capable of accelerating ECM
From human peripheral blood, monocytes can be differentiated into uncommitted macrophages (M0) and then polarized to M1 and M2 phenotypes by potent hematopoietic growth factors such as granulocyte-macrophage colony stimulating factor (GM-CSF) and macrophage stimulating factor (M-CSF), respectively . On one hand, M1 is usually induced by microbial stimuli, such as lipopolysaccharide (LPS), and pro-inflammatory cytokines, as interferon-ᵞ (IFN-ᵞ) and tumor necrosis factor-α (TNF-α), promoting a pro-inflammatory response responsible for extracellular matrix degradation and tissue injury. On the other hand, M2 macrophages are stimulated by interleukin (IL)-4 and/or IL-13, producing transforming growth factor beta (TGF-β) and IL-10, that are usually involved in inflammatory response resolution and wound healing, promoting extracellular matrix construction and cell proliferation with a characteristic anti-inflammatory response .
are key participants in the acute inflammatory features of RA . Nevertheless, increasing evidence suggests that a variety of innate effector cells, such as monocytes and macrophages, are also closely involved in the development of synovial inflammation in RA. In fact, massive infiltration of activated monocytes/macrophages are frequently observed in synovial membranes of RA patients. These are a major source of cytokines (such as TNF-, IL-1β, IL-8, and GM-CSF) in the inflamed joints . The initial milestone for RA pathogenesis onset is thought to be the activation of the innate immune response, including the activation of dendritic cells by exogenous material and autologous antigens . Antigen-presenting cells, including dendritic cells, macrophages and activated B cells, present arthritis-associated antigens to T cells [35,39]. These T cells are upregulated by various lymphokines, including interleukin 2 (IL-2) and interferon γ (IFNγ). Upon stimulation, T cells induce activation of macrophages, B cells, fibroblasts, and osteoclasts [38,39]. In turn, B lymphocytes express various cell-surface molecules, including antigen receptor immunoglobulin and differentiation antigens, such as CD20 and CD22. Upon differentiation, they turn into plasma cells that secrete antibodies, including autoantibodies to IgG (rheumatoid factor), to citrullinated peptides such as vimentin, fibrinogen, or cyclic citrullinated peptide (CCP) and to rheumatoid arthritis antigen (RA33) [42,43]. On the other hand, the formation of immune complexes by autoantibodies can increase the production of proinflammatory cytokines such as TNF-. In fact, the occurrence of autoantibodies RF and anti-CCP are associated with severe rheumatoid arthritis and can be used as an effective strategy for identifying patients with RA at high risk for poor outcome . When activated, B cells also serve as APCs, inducing T cell activation, and potentially leading to the perpetuation of the immune-mediated proinflammatory response . Besides cells of the innate and adaptive immune system, several other inflammatory cell populations infiltrate the synovial membrane in rheumatoid arthritis patients [34,35,45].
during pathogenic infection or tissue injury and to contribute to the maintenance of tissue homeostasis (1–3). Macrophages were first identified in the late 19th century by Élie Metchnikoff (1845–1916) and designated as large phagocytes (4, 5). Based on their phagocytic activity, macrophages were first classified as cells from the reticuloendothelial system, which also comprised endothelial cells, fibroblasts, spleen and lymphoid reticular cells, Kupffer cells, splenocytes, and monocytes (6). However, because endocytosis performed by endothelial cells is a process that is distinct from phagocytosis, by the late 1960s a new classification system for mononuclear phagocytic cells as cells from “mononu- clear phagocytic system” (MPS) was proposed (7). The MPS was defined as a group of phagocytic cells sharing morphological and functional similarities, including pro-monocytes, monocytes, macrophages, dendritic cells (DCs), and their bone marrow (BM) progenitors (7–12). Although the phagocytic cells play similar roles in orchestrating the immune response and maintaining tis- sue homeostasis (11), they represent cell populations that are extremely heterogeneous (13), and the general classification of mononuclear cells in a unique system is currently under intense discussion (12, 14). In this context, Guilliams et al. suggested a classification of MPS cells based primarily on their ontogeny and secondary on their location, function, and phenotype, promoting a better classification under both steady state and inflammatory conditions (14).
Macrophages have long been implicated as central participants in the process of angiogenesis [25,26]. Both as a source of endothelial cell growth factors , as well as matrix metallopro- teinases essential to the invasion of new vessels , macrophages have been shown to be abundant at the sites of neovascularization and critical to the process. Previous work from our laboratory showed a significant positive correlation between the number of macrophages in the bronchoalveolar lavage fluid of the left lung after complete pulmonary ischemia and the extent of systemic angiogenesis . Furthermore, macrophage-derived growth factors, the CXC chemokines, are upregulated in the ischemic lung  and are essential to the process of neovascularization [10,11]. In the present study, we sought to better characterize the trapped monocytes/macrophages in the lung early after the onset of pulmonary ischemia. Although, previous studies have shown monocyte differentiation in lung injury models and the recruit- ment of specific macrophage subtypes, our model offered an opportunity to study changes in monocyte phenotype in a closed, in vivo environment where no new cells could be recruited until 5– 7 days after LPAL when new vessels were formed. We hypo- thesized that monocytes both differentiate and proliferate in response to sustained pulmonary ischemia and contribute to lung remodeling through subsequent systemic angiogenesis. Our experimental results demonstrate an increase in the number of mature lung macrophages early after the onset of ischemia and suggest an essential nature of these cells for angiogenesis.
Arachidonic acid (AA), a normal component of cell membrane phospholipids, is a substrate for prostaglandin endoperoxide (PGH) syntases-1 and -2, also known as cyclooxygenase (COX)-1 and -2, lipoxygenase (5-, 12-, or 15) (LO) or cytochrome p450 enzymes. Leukotrienes are produced in a multi-step enzyme pathway called the 5-lipoxygenase (5-LO) pathway, which is active in leukocytes such as neutrophils, eosinophils, mast cells and monocytes . LTs exert their biological effects by activating specific receptors belonging to the superfamily of G protein-coupled receptors, including both LTB 4 and the cysteinyl LTs (cys-LTs) C 4 , D 4 , and E 4 . Two receptors for LTB 4 have been identified: BLT1 and BLT2. BLT1 is a high-affinity receptor that is specific for LTB 4 , which is expressed primarily in leukocytes and mediates chemotaxis; BLT2 is a pharmacologically distinct receptor that is ubiquitously expressed, displays a low affinity for LTB4 and binds to other eicosanoids. Receptors that are activated in response to Cys-LTs were cloned in 1999 and termed CysLT1 and CysLT2. CysLT1 recombinant receptor is activated by all of the native ligands with the following order of potency: LTD4 > LTC4 > LTE4. In contrast, the agonist rank order potency for the CysLT2 receptor is LTD 4 = LTC 4 , with LTE 4 demonstrating less potency .
Differentiated macrophages infected by HIV in vitro are more resistant to TRAIL-mediated cell death triggered by the envelope protein  whereas another report suggests that HIV-infected macrophages are more prone to undergo apoptosis . In the peripheral blood of chronically HIV-infected individuals and SIV- infected rhesus macaques (RMs), reduced numbers of DCs are found [55,56,57,58,59,60,61] consistent with increased death of those cells [62,63,64]. Furthermore, in chronically SIV-infected RMs, massive turnover of peripheral monocytes undergoing apoptosis have been reported . In viremic HIV-infected individuals it has been shown that both spontaneous and IFN-a˜- induced monocyte cell death are elevated compared to controls  although another report describes monocytes resistant to cell death, associated with antiapoptotic gene profiles . However, little information exists on the precise molecular mechanisms involved and only few studies have assessed these processes early after infection.
TNF- 𝛼 and IL-10 production by such cells was inhibited by high concentrations of Ci, while an increased fungicidal activity was seen against C. albicans. Although TNF- 𝛼 is important to activate monocytes and macrophages while IL-10 may suppress these cells, one may speculate that the fungicidal activity of monocytes incubated with Ci may have involved other effector mechanisms, such as nitrogen and oxygen reactive intermediates after interaction with the fungus. It has been reported that hydrogen peroxide (H 2 O 2 ) could be efficient in the first hours after phagocytosis, while nitric oxide (NO) could be important either in the beginning or late in the antimicrobial activity of macrophages . Although these mediators were not determined herein, the highest concentrations of Ci could have induced both H 2 O 2 and NO, increasing the fungicidal activity of monocytes.
two main mechanisms occur at steady-state to maintain the pool of these cells. The first one is self-renewal and is observed mainly in the heart, brain and kidney (Varol et al., 2015). However, as the host ages or is subjected to tissue injury, circulating monocytes can transdifferentiate into functional tissue-resident macrophages (Perdiguero and Geissmann, 2015). Having established that the expression of FTH by macrophages is essential to restore survival of R26.Fth Δ/Δ mice (Figure 3.4B), the question arose if expression of FTH in either circulating monocytes or tissue resident macrophages was responsible for this phenomenon. We used for that purpose Ccr2 deficient (Ccr2 -/- ) mice in which the absence of this chemokine receptor prevents monocyte migration from the bone marrow into parenchymal tissues (Boring et al., 1997; Varol et al., 2015). We assumed that bone marrow macrophages from Ccr2 -/- presumably still express normal levels of ferritin that upon secretion could possibly sustain the survival of R26.Fth Δ/Δ recipient mice reconstituted with Ccr2 -/- bone marrow, i.e. Ccr2 -/- R26.Fth Δ/Δ mice. Upon deletion of the Fth
The tumor microenvironment plays a critical role in shaping TAM activation, as functional skewing of mononuclear phagocytes occurs in vivo. Differentiation of monocytes in the unique cytokine milieu of the tumor site polarizes resulting macrophages into M1 (immuno-stimula- tory) or M2 (immuno-suppressive) TAMs. In non-progressing or regressing tumors, TAMs resemble classically activated M1 macrophages, as they produce pro-inflammatory cytokines, demonstrate enhanced antigen presentation, and mediate tumor lysis . In contrast, TAMs assume an alternatively activated M2 activation state in malignant tumors, as they suppress adaptive immune responses and secrete anti-inflammatory mediators and angiogenic factors that support tumor growth and metastasis . Because macrophage phenotype and function are plastic and may be redirected by immuno-modulatory cues , re-programming TAM polarization from M2 to M1 may have significant therapeutic benefit. While many attempts have been made to redirect TAM activation using cytokines and immune-activators such as LPS , these approaches have not been successful for many reasons, including issues with delivery and systemic toxicity. To the best of our knowledge, no drugs have been identified for the treatment of breast cancer that repolarize TAMs.
polymorphonuclear cell recruitment to the site of infec- tion, and neutrophils have been shown to act as "Trojan horses"  for Leishmania establishment. Indeed, devel- opment of clinically evident lesions occurs in parallel with the influx of inflammatory cells, including neutrophils, eosinophils and macrophages . Using the air pouch model of inflammation, we observed a significant increase in the recruitment of polymorphonuclear cells upon injection of L. intermedia SGS (de Moura et al., man- uscript in preparation). In mice exposed to L. intermedia SGS, a further inoculation of L. intermedia SGS induced an important inflammatory response comprised of numer- ous polymorphonuclear and few mononuclear cells . We can thus hypothesize that the increase in IL-8 produc- tion induced by L. intermedia saliva may lead to neu- trophil recruitment and, consequently, the successful establishment of infection. Moreover, it will also be inter- esting to determine whether L. intermedia saliva also exerts effects on the effector functions of T-lymphocytes, employing, for example in vitro priming systems [30,14].
- phage apoptosis in TB have been the subject of intense investigation and debate. Apoptosis, but not necrosis of infected monocytes is coupled with killing of intracellular BCG [50,51]. Although all mycobacteria are capable of inducing apoptosis to some degree, less virulent mycobacteria such as BCG and avirulent Mtb (H37Ra) are better inducers of macrophage apoptosis when directly compared to the more virulent mycobac- teria such as Mtb (H37Rv)[11,33,37,52]. From these data a hypothesis has emerged that mycobacterial pathogenicity is inversely correlated with the ability of macrophages to undergo apoptosis . This hypothesis is supported by results that suggest two distinct advantages in promoting macrophage apoptosis during early mycobacterial infection. First, the increased bacterial killing that was observed in apoptotic macrophages in combination with clearance of apoptotic cells may prevent the spread of infection and lead to sterilization of the infected host [12,39,51,53,54]. Second, the uptake of infected apoptotic macrophages by dendritic cells (DCs) leads to the breakdown
(,50 copies/ml) to unsuppressed (.800,000 RNA copies/ml). This finding was consistent with our previous microarray study  which suggested that viral load was a determining factor in Sn expression. This interpretation was further supported by data from subjects whose viral loads, initially high, were suppressed to undetectable levels following HAART treatment and exhibited a concurrent decrease in Sn expression. In combination with our in vitro data showing that IFN-a and IFN-c induce Sn expression in cultured human monocytes and THP-1 cells, it is possible that either of these cytokines drives Sn expression during HIV-1 infection. IFN-c, which is associated with immune activation, is produced by T cells and NK cells during the acute period of infection . By comparison, IFN-a, which is present in the serum of individuals infected with HIV-1 [28,29], is released by plasmacytoid dendritic cells in an innate antiviral response [30,31]. In a study by Tilton et al., monocyte production of proinflammatory cytokines, IL-1b, IL-6 and TNF-a was dimin- ished in HIV viremia suggesting that monocyte function was impaired during unsuppressed viral replication . Coincident with the loss of monocyte function was an increase in Sn expression. Importantly, this particular phenotype could be recapitulated by stimulating monocytes with IFN-a . Further substantiating the link between Sn expression and viral load was an observation in a treatment naı¨ve population that Sn mRNA expression in CD14+ monocytes dramatically increased shortly after HIV-1 infection and continued to rise in patients who progressed to AIDS . Since high viral load is a common feature of AIDS, elevated Sn expression would be predicted in these patients. In aggregate, our data suggest that elevated Sn expression on monocytes is consistent with an antiviral response, as opposed to a restricted marker of inflammation observed in tissue macrophages associated with rheumatoid arthritis . While our findings indicate that either IFN-a or IFN-c could induce Sn expression in peripheral monocytes, determining the specific conditions and cytokines responsible for Sn induction will require additional research.
It is also possible that CD14 is required for early uptake of ligands at low concentration by clathrin-dependent endocytosis, while other regulators may contribute to late uptake of ligands at higher concentrations. In support of this hypothesis, it was shown that TLRβ signaling may vary depending on ligand concentration [γ4] and that CD14 is required for recognition of low concentrations of LPS [γ5]. Furthermore, Zanoni et al. have shown that while CD14 is important for TLR4-MyD88- dependent signal transduction only at low concentrations of LPS, the function of CD14 is independent of the signaling triggered by TLR4 obtained at high LPS concentrations . Likewise, while MyD88 was shown to be critical for TNF production in macrophages in response to S.Aureus, LTA and Pam γ CSK 4 , it did not play a significant role in pathogen
APC dysfunction could be a significant factor in immune dysfunction since both cell mediated and humoral immunity rely on APC function to activate their specific responses. There have been several in vitro studies where macrophages are infected with HIV or transfected to express specific HIV molecules such as Nef and Vpu [2,12,13,14]. These studies have demonstrated multiple inhibitory effects on APC function by HIV virions or specific HIV products. These effects include inhibition of MHC-II expression, increase in invariant chain expression, decrease in antigen uptake, and decreased antigen presentation overall. These studies though all have a similar feature in their experimental systems of very high levels of HIV-infection or expression of a transfected HIV molecule. This is not the case in vivo where the frequency of HIV-infected MN and macrophages is very low. In addition it is well known that MN are resistant to becoming productively infected due to a pre-integration block . Both of these features suggest that APC dysfunction if present would be due to bystander effects rather than direct infection. We did not detect a defect in either antigen processing or presentation by MN isolated from HIV+ individuals. Our results using a very specific measurement tool of MHC-II antigen processing and presentation are similar to the observations reported by Blauvelt et al. [5,6].
SLAMF7 was originally identified as a NK cell-associated surface molecule . Subsequently, it was shown to be expressed on lymphocytes and monocytes . More recently, a reduced expression on monocytes and NK cells with a simultaneous increase of SLAMF7 on B cells was observed in patients with lupus erythematosus . The strongest link to SLAMF7 as an M1 marker gene comes from observations in intestine allograft rejection, demonstrating that tissue macrophages derived from patients rejecting the graft showed elevated levels of SLAMF7 . It would be interesting to see if macrophages in other settings of transplant rejection are also enriched for this novel M1 marker gene. Considering the identification of single specific marker genes for macrophage polarization our findings clearly point to the necessity for multi-parameter analysis instead. This can be exemplified by the differential expression of CD1a and CD1b, two cell surface molecules that are mainly studied in context of antigen presentation by dendritic cells . Previous reports suggested upregulation of CD1 proteins on human monocytes by GM-CSF . However, we clearly present evidence that expression is induced in both M-CSF and GM-CSF driven macrophages and polarization towards M2-like macrophages is significantly increasing expression of CD1a and CD1b suggesting that they might be up-regulated on tissue macrophages in an M2- driving environment. This is similarly true for CD93, which was originally identified to be expressed on early hematopoietic stem cells and B cells . CD93 is involved in biological processes such as adhesion, migration, and phagocytosis [52,53]. CD93 expressed on myeloid cells can be shed from the cell surface and the soluble form seems to be involved in differentiation of monocytes towards a macrophage phenotype . Since soluble CD93 has been implicated in inflammatory responses, it will be important to further elucidate how polarization-induced differential expression of CD93 contributes to specific inflammatory responses. Another surprising finding is the differential expression of CD226 between human M1- and M2-like macrophages, a molecule initially shown to be involved in cytolytic function of T cells . Subsequently, it could be shown that CD226 has additional functions including the regulation of monocyte migration through endothelial junctions . Similar to the other M2-associated markers, so far little is known about CD226 on polarized macrophages. Since CD226 expression levels on lymphocytes have been implicated in
One of the kinases affected by Leishmania is Janus kinase 2 (JAK 2), a member of the Janus family of tyrosine kinases that play an important role in immune activation. The JAK signaling pathway starts when a cytokine or a growth factor binds to its receptor inducing a signaling cascade that ultimately results in the phosphorylation of a signal transducer, an activator transcription (STAT) and a transcription factor (TF) that consequently leading to the activation or repression of gene transcription (62). The alteration of the JAK signaling is advantageous for the Leishmania because the iNOS gene promoter has binding sites for several TFs and the parasite has the ability to block the JAK/STAT signaling pathway in response to the IFN-γ stimulation, thus decreasing NO production by macrophages (63). Nandan and Reiner reported that infection with L. donovani amastigotes was able to block IFN-γ-induced JAK1, JAK2, and STAT-1 phosphorylation in PMA differentiated U-937 promonocytic cells and human monocytes (64). Another study demonstrated that L. donovani promastigotes rapidly activate host SHP-1 leading to the subsequent inhibition of IFN-γ-induced JAK2 phosphorylation (65). The mitogen-activated protein (MAP) kinases family also seems to be modulated by Leishmania parasites. The MAP kinases are serine/threonine kinases that link transmembrane signaling with gene transcription events and are divided in three major subgroups: the extracellular signal-regulated kinases (ERKs); the p38 MAP kinase and the c-jun amino-terminal kinases (JNKs). It has been reported that synthetic Leishmania LPGs activate ERK MAP kinase in macrophages which suppress the transcription of IL-12p40 (66). Another kinase affected by the parasite is protein kinase C, which activity was shown to be decreased by Leishmania LPG (67).
Monocytes differentiate into heterogeneous populations of tissue macrophages and dendritic cells (DCs) that regulate inflammation and immunity. Identifying specific populations of myeloid cells in vivo is problematic, however, because only a limited number of proteins have been used to assign cellular phenotype. Using mass spectrometry and bone marrow- derived cells, we provided a global view of the proteomes of M-CSF-derived macrophages, classically and alternatively activated macrophages, and GM-CSF-derived DCs. Remarkably, the expression levels of half the plasma membrane proteins differed significantly in the various populations of cells derived in vitro. Moreover, the membrane proteomes of macrophages and DCs were more distinct than those of classically and alternatively activated macrophages. Hierarchical cluster and dual statistical analyses demonstrated that each cell type exhibited a robust proteomic signature that was unique. To interrogate the phenotype of myeloid cells in vivo, we subjected elicited peritoneal macrophages harvested from wild-type and GM-CSF-deficient mice to mass spectrometric and functional analysis. Unexpectedly, we found that peritoneal macrophages exhibited many features of the DCs generated in vitro. These findings demonstrate that global analysis of the membrane proteome can help define immune cell phenotypes in vivo.
assumption that PIs and/or dyslipidemia are the primary source of development of atherosclerosis in HIV patients. Results presented in this report suggest that HIV-induced impairment of cholesterol efﬂux from macrophages may be another important contributor to the pathogenesis of CAD. Indeed, inactivation of ABCA1 in macrophages of hyper- lipidemic mice signiﬁcantly increased development of athe- rosclerosis , and genetic mutation inactivating ABCA1 in humans leads to Tangier disease, one of the characteristic features of which is an increased risk of CAD . Impair- ment of reverse cholesterol transport mediated by down- regulation of ABCA1 has been described for bacterial infections and has been linked to pathogenesis of athero- sclerosis (reviewed in ). In the case of HIV infection, this mechanism would have only a mild atherogenic effect or not at all on the background of hypocholesterolemia character- istic for untreated HIV-1 infection [55,57]. Treatment of HIV- infected patients with HAART causes a sharp rise of triglyceride-rich VLDL, resulting in enhanced lipid uptake and foam cell formation , and small dense LDL [59,60], which is particularly susceptible to oxidation , is more able to inﬁltrate the subendothelial space, and is a risk factor for CAD . A combination of these effects of HAART and impairment of cholesterol efﬂux by HIV (which prevents compensatory removal of excessive cholesterol) would result in a greatly enhanced accumulation of cholesterol in HIV- infected macrophages and would potentially further increase the risk of development of atherosclerosis. It should be noted that HIV-infected macrophages, unlike T cells, survive for extended periods of time and are considered long-term reservoirs of HIV-1 . As a result, infected macrophages persist, at least for some time, in HAART-treated patients, when conditions favor development of atherosclerotic pla- ques. We can speculate that these macrophages may contrib- ute to initiation of atherosclerotic plaque formation, which then proceeds even in the absence of newly infected cells. This mechanism is consistent with the presence of HIV- infected macrophages in atherosclerotic plaques of HAART- treated patients observed in our study (Figure 8). However, further in vivo and clinical studies are required to evaluate the contribution of the impairment of reverse cholesterol transport to the risk of atherosclerosis in HIV patients.
phages. To gain an in-depth knowledge of the mechanisms by which D -LFcin17–30 inhibits mycobacterial growth, transmission electron microscopy (TEM) was performed on M. avium-infected macrophages treated with the lactoferricin peptides. Represen- tative images of these assays are shown in Fig. 4. Striking alterations in macrophage ultrastructure were evident when they were treated with D -LFcin17–30 (Fig. 4C). As was expected, intact mycobacteria were difﬁcult to detect, whereas in nontreated macro- phages or even in LFcin17–30-treated macrophages, intact mycobacteria were visual- ized (Fig 4A and B, arrowheads). Several double-membrane vesicles containing di- gested material, suggestive of autophagosomes (Fig. 4C, asterisks), were observed in D -LFcin17–30-treated macrophages. A high number of dense vesicles and multivesicu- lar bodies loaded with dense material were also seen (Fig. 4C, black arrows). Large structures, exhibiting several membranes and delimitations inside, suggestive of cell material ingestion and fusion with endosomes, lysosomes, or autophagosomes (Fig. 4C, white arrows), were frequently seen. These alterations were not observed in the case of cells treated with LFcin17–30 (Fig. 4B). These observations suggested that D -LFcin17–30 induced signiﬁcant alterations in the macrophage vesicular trafﬁc and membrane digestion pathways, which might contribute to mycobacterial killing.