Absence of Bim sensitizes mice to experimental Trypanosoma cruzi infection.

Chagas disease is a life-threatening disorder caused by the protozoan parasite Trypanosoma cruzi. Parasite-specific antibodies, CD8+ T cells, as well as IFN-γ and nitric oxide (NO) are key elements of the adaptive and innate immunity against the extracellular and intracellular forms of the parasite. Bim is a potent pro-apoptotic member of the Bcl-2 family implicated in different aspects of the immune regulation, such as negative selection of self-reactive thymocytes and elimination of antigen-specific T cells at the end of an immune response. Interestingly, the role of Bim during infections remains largely unidentified. To explore the role of Bim in Chagas disease, we infected WT, Bim+/-, Bim-/- mice with trypomastigotes forms of the Y strain of T. cruzi. Strikingly, our data revealed that Bim-/- mice exhibit a delay in the development of parasitemia followed by a deficiency in the control of parasite load in the bloodstream and a decreased survival compared to WT and Bim+/- mice. At the peak of parasitemia, peritoneal macrophages of Bim-/- mice exhibit decreased NO production, which correlated with a decrease in the pro-inflammatory Small Peritoneal Macrophage (SPM) subset. A similar reduction in NO secretion, as well as in the pro-inflammatory cytokines IFN-γ and IL-6, was also observed in Bim-/- splenocytes. Moreover, an impaired anti-T. cruzi CD8+ T-cell response was found in Bim-/- mice at this time point. Taken together, our results suggest that these alterations may contribute to the establishment of a delayed yet enlarged parasitic load observed at day 9 after infection of Bim-/- mice and place Bim as an important protein in the control of T. cruzi infections.

IL-1α promotes liver inflammation and necrosis during blood-stage Plasmodium chabaudi malaria.

Malaria causes hepatic inflammation and damage, which contribute to disease severity. The pro-inflammatory cytokine interleukin (IL)-1α is released by non-hematopoietic or hematopoietic cells during liver injury. This study established the role of IL-1α in the liver pathology caused by blood-stage P. chabaudi malaria. During acute infection, hepatic inflammation and necrosis were accompanied by NLRP3 inflammasome-independent IL-1α production. Systemically, IL-1α deficiency attenuated weight loss and hypothermia but had minor effects on parasitemia control. In the liver, the absence of IL-1α reduced the number of TUNEL+ cells and necrotic lesions. This finding was associated with a lower inflammatory response, including TNF-α production. The main source of IL-1α in the liver of infected mice was inflammatory cells, particularly neutrophils. The implication of IL-1α in liver inflammation and necrosis caused by P. chabaudi infection, as well as in weight loss and hypothermia, opens up new perspectives for improving malaria outcomes by inhibiting IL-1 signaling.

P2X7 receptor drives Th1 cell differentiation and controls the follicular helper T cell population to protect against Plasmodium chabaudi malaria.

A complete understanding of the mechanisms underlying the acquisition of protective immunity is crucial to improve vaccine strategies to eradicate malaria. However, it is still unclear whether recognition of damage signals influences the immune response to Plasmodium infection. Adenosine triphosphate (ATP) accumulates in infected erythrocytes and is released into the extracellular milieu through ion channels in the erythrocyte membrane or upon erythrocyte rupture. The P2X7 receptor senses extracellular ATP and induces CD4 T cell activation and death. Here we show that P2X7 receptor promotes T helper 1 (Th1) cell differentiation to the detriment of follicular T helper (Tfh) cells during blood-stage Plasmodium chabaudi malaria. The P2X7 receptor was activated in CD4 T cells following the rupture of infected erythrocytes and these cells became highly responsive to ATP during acute infection. Moreover, mice lacking the P2X7 receptor had increased susceptibility to infection, which correlated with impaired Th1 cell differentiation. Accordingly, IL-2 and IFNγ secretion, as well as T-bet expression, critically depended on P2X7 signaling in CD4 T cells. Additionally, P2X7 receptor controlled the splenic Tfh cell population in infected mice by promoting apoptotic-like cell death. Finally, the P2X7 receptor was required to generate a balanced Th1/Tfh cell population with an improved ability to transfer parasite protection to CD4-deficient mice. This study provides a new insight into malaria immunology by showing the importance of P2X7 receptor in controlling the fine-tuning between Th1 and Tfh cell differentiation during P. chabaudi infection and thus in disease outcome.

F4/80 as a Major Macrophage Marker: The Case of the Peritoneum and Spleen.

Tissue macrophages are a heterogeneous cell population residing in all body tissues that contribute to the maintenance of homeostasis and trigger immune activation in response to injurious stimuli. This heterogeneity may be associated with tissue-specific functions; however, the presence of distinct macrophage populations within the same microenvironment indicates that macrophage heterogeneity may also be influenced outside of tissue specialization. The F4/80 molecule was established as a unique marker of murine macrophages when a monoclonal antibody was found to recognize an antigen exclusively expressed by these cells. However, recent research has shown that F4/80 is expressed by other immune cells and is not equivalently expressed across tissue-specific macrophage lineages, including those residing in the same microenvironment, such as the peritoneum and spleen. In this context, two murine macrophage subtypes with distinct F4/80 expression patterns were recently found to coexist in the peritoneum, termed large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs). However, the presence of phenotypic and functional heterogeneous macrophage subpopulations in the spleen was already known. Thus, although F4/80 surface expression continues to be the best method to identify tissue macrophages, additional molecules must also be examined to distinguish these cells from other immune cells.

P2X7 Receptor in Bone Marrow-Derived Cells Aggravates Tuberculosis Caused by Hypervirulent Mycobacterium bovis.

Tuberculosis (TB) remains a serious public health problem despite the great scientific advances in the recent decades. We have previously shown that aggressive forms of TB caused by hypervirulent strains of Mycobacterium tuberculosis and Mycobacterium bovis are attenuated in mice lacking the P2X7 receptor, an ion channel activated by extracellular ATP. Therefore, P2X7 receptor is a potential target for therapeutic intervention. In vitro, hypervirulent mycobacteria cause macrophage death by a P2X7-dependent mechanism that facilitates bacillus dissemination. However, as P2X7 receptor is expressed in both bone marrow (BM)-derived cells and lung structural cells, several cellular mechanisms can operate in vivo. To investigate whether the presence of P2X7 receptor in BM-derived cells contributes to TB severity, we generated chimeric mice by adoptive transfer of hematopoietic cells from C57BL/6 or P2X7-/- mice into CD45.1 irradiated mice. After infection with hypervirulent mycobacteria (MP287/03 strain of M. bovis), P2X7-/->CD45.1 mice recapitulated the TB resistance observed in P2X7-/- mice. These chimeric mice showed lower lung bacterial load and attenuated pneumonia compared to C57BL/6>CD45.1 mice. Lung necrosis and bacterial dissemination to the spleen and liver were also reduced in P2X7-/->CD45.1 mice compared to C57BL/6>CD45.1 mice. Furthermore, an immature-like myeloid cell population showing a Ly6Gint phenotype was observed in the lungs of infected C57BL/6 and C57BL/6>CD45.1 mice, whereas P2X7-/- and P2X7-/->CD45.1 mice showed a typical neutrophil (Ly6Ghi) population. This study clearly demonstrates that P2X7 receptor in BM-derived cells plays a critical role in the progression of severe TB.

P2X7 receptor in bone marrow-derived cells aggravates tuberculosis caused by hypervirulent mycobacterium bovi

Tuberculosis (TB) remains a serious public health problem despite the great scientific advances in the recent decades. We have previously shown that aggressive forms of TB caused by hypervirulent strains of Mycobacterium tuberculosis and Mycobacterium bovis are attenuated in mice lacking the P2X7 receptor, an ion channel activated by extracellular ATP. Therefore, P2X7 receptor is a potential target for therapeutic intervention. In vitro, hypervirulent mycobacteria cause macrophage death by a P2X7-dependent mechanism that facilitates bacillus dissemination. However, as P2X7 receptor is expressed in both bone marrow (BM)-derived cells and lung structural cells, several cellular mechanisms can operate in vivo. To investigate whether the presence of P2X7 receptor in BM-derived cells contributes to TB severity, we generated chimeric mice by adoptive transfer of hematopoietic cells from C57BL/6 or P2X7-/- mice into CD45.1 irradiated mice. After infection with hypervirulent mycobacteria (MP287/03 strain of M. bovis), P2X7-/->CD45.1 mice recapitulated the TB resistance observed in P2X7-/- mice. These chimeric mice showed lower lung bacterial load and attenuated pneumonia compared to C57BL/6>CD45.1 mice. Lung necrosis and bacterial dissemination to the spleen and liver were also reduced in P2X7-/->CD45.1 mice compared to C57BL/6>CD45.1 mice...

Splenic Macrophage Subsets and Their Function during Blood-Borne Infections.

The spleen is one of the major immunological sites for maintaining blood homeostasis. Previous studies showed that heterogeneous splenic macrophage populations contribute in complimentary ways to control blood-borne infections and induce effective immune responses. Marginal metallophilic macrophages (MMMΦs) and marginal zone macrophages (MZMΦs) are cells with great ability to internalize blood-borne pathogens such as virus or bacteria. Their localization adjacent to T- and B-cell-rich splenic areas favors the rapid contact between these macrophages and cells from adaptive immunity. Indeed, MMMΦs and MZMΦs are considered important bridges between innate and adaptive immunity. Although red pulp macrophages (RpMΦs) are mainly considered scavengers for senescent erythrocytes, several data indicate a role for RpMΦs in control of infections such as blood-stage malaria as well as in the induction of innate and adaptive immunity. Here, we review current data on how different macrophage subsets recognize and help eliminate blood-borne pathogens, and, in turn, how the inflammatory microenvironment in different phases of infection (acute, chronic, and after pathogen clearance) influences macrophage function and survival.

Revisiting mouse peritoneal macrophages: heterogeneity, development, and function.

Tissue macrophages play a crucial role in the maintenance of tissue homeostasis and also contribute to inflammatory and reparatory responses during pathogenic infection and tissue injury. The high heterogeneity of these macrophages is consistent with their adaptation to distinct tissue environments and specialization to develop niche-specific functions. Although peritoneal macrophages are one of the best-studied macrophage populations, recently it was demonstrated the co-existence of two subsets in mouse peritoneal cavity (PerC), which exhibit distinct phenotypes, functions, and origins. These macrophage subsets have been classified, according to their morphology, as large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs). LPMs, the most abundant subset under steady state conditions, express high levels of F4/80 and low levels of class II molecules of the major histocompatibility complex (MHC). LPMs appear to be originated from embryogenic precursors, and their maintenance in PerC is regulated by expression of specific transcription factors and tissue-derived signals. Conversely, SPMs, a minor subset in unstimulated PerC, have a F4/80(low)MHC-II(high) phenotype and are generated from bone-marrow-derived myeloid precursors. In response to infectious or inflammatory stimuli, the cellular composition of PerC is dramatically altered, where LPMs disappear and SPMs become the prevalent population together with their precursor, the inflammatory monocyte. SPMs appear to be the major source of inflammatory mediators in PerC during infection, whereas LPMs contribute for gut-associated lymphoid tissue-independent and retinoic acid-dependent IgA production by peritoneal B-1 cells. In the previous years, considerable efforts have been made to broaden our understanding of LPM and SPM origin, transcriptional regulation, and functional profile. This review addresses these issues, focusing on the impact of tissue-derived signals and external stimulation in the complex dynamics of peritoneal macrophage populations.

In vivo approaches reveal a key role for DCs in CD4+ T cell activation and parasite clearance during the acute phase of experimental blood-stage malaria.

Dendritic cells (DCs) are phagocytes that are highly specialized for antigen presentation. Heterogeneous populations of macrophages and DCs form a phagocyte network inside the red pulp (RP) of the spleen, which is a major site for the control of blood-borne infections such as malaria. However, the dynamics of splenic DCs during Plasmodium infections are poorly understood, limiting our knowledge regarding their protective role in malaria. Here, we used in vivo experimental approaches that enabled us to deplete or visualize DCs in order to clarify these issues. To elucidate the roles of DCs and marginal zone macrophages in the protection against blood-stage malaria, we infected DTx (diphtheria toxin)-treated C57BL/6.CD11c-DTR mice, as well as C57BL/6 mice treated with low doses of clodronate liposomes (ClLip), with Plasmodium chabaudi AS (Pc) parasites. The first evidence suggesting that DCs could contribute directly to parasite clearance was an early effect of the DTx treatment, but not of the ClLip treatment, in parasitemia control. DCs were also required for CD4+ T cell responses during infection. The phagocytosis of infected red blood cells (iRBCs) by splenic DCs was analyzed by confocal intravital microscopy, as well as by flow cytometry and immunofluorescence, at three distinct phases of Pc malaria: at the first encounter, at pre-crisis concomitant with parasitemia growth and at crisis when the parasitemia decline coincides with spleen closure. In vivo and ex vivo imaging of the spleen revealed that DCs actively phagocytize iRBCs and interact with CD4+ T cells both in T cell-rich areas and in the RP. Subcapsular RP DCs were highly efficient in the recognition and capture of iRBCs during pre-crisis, while complete DC maturation was only achieved during crisis. These findings indicate that, beyond their classical role in antigen presentation, DCs also contribute to the direct elimination of iRBCs during acute Plasmodium infection.

Role of interplay between IL-4 and IFN-γ in the in regulating M1 macrophage polarization induced by Nattectin.

Recently our group described that Nattectin, a C-type lectin of the venom of Thalassophryne nattereri shows a potent pro-inflammatory capacity. Here, we demonstrated that Nattectin is able to induce M1 macrophage marker iNOS, and up-regulate the expression of MHC class II, CD80, CD86 and CD40 molecules. The increase in MHC class II and CD49a integrin expression with MMP-9 production and endocytic capacity depend on lectin function of Nattectin. Moreover, the polarization of peritoneal and bone marrow-derived macrophages induced by Nattectin to M1 profile is dependent on Th1 cytokines (IL-12 and IFN-γ), and negatively regulated by Th2 cytokines (IL-4, IL-10 and IL-13). Also we reveal that IL-4 play a dual role in this polarization: a regular action of IL-4 was seen in the negative regulation of the CD40 expression, but an unexpected positive regulation was seen in the expression of CCR7 and MHC class II. Finally, our in vivo studies showed that the influx of neutrophils and small peritoneal macrophage--F4/80(low)MHCII(hi) induced by Nattectin is totally dependent on IL-4 and IFN-γ cytokines. Furthermore, the induction of IL-6 release is negatively regulated by IL-4 and positively regulated by IL-12 and IFN-γ. Together, the results allowed us to expand the knowledge about the regulation of macrophage activation, as well as confirmed the ability of Nattectin, a fish C-type lectin, as an important immunomodulatory agent.

Cellular renewal and improvement of local cell effector activity in peritoneal cavity in response to infectious stimuli.

The peritoneal cavity (PerC) is a singular compartment where many cell populations reside and interact. Despite the widely adopted experimental approach of intraperitoneal (i.p.) inoculation, little is known about the behavior of the different cell populations within the PerC. To evaluate the dynamics of peritoneal macrophage (MØ) subsets, namely small peritoneal MØ (SPM) and large peritoneal MØ (LPM), in response to infectious stimuli, C57BL/6 mice were injected i.p. with zymosan or Trypanosoma cruzi. These conditions resulted in the marked modification of the PerC myelo-monocytic compartment characterized by the disappearance of LPM and the accumulation of SPM and monocytes. In parallel, adherent cells isolated from stimulated PerC displayed reduced staining for ß-galactosidase, a biomarker for senescence. Further, the adherent cells showed increased nitric oxide (NO) and higher frequency of IL-12-producing cells in response to subsequent LPS and IFN-γ stimulation. Among myelo-monocytic cells, SPM rather than LPM or monocytes, appear to be the central effectors of the activated PerC; they display higher phagocytic activity and are the main source of IL-12. Thus, our data provide a first demonstration of the consequences of the dynamics between peritoneal MØ subpopulations by showing that substitution of LPM by a robust SPM and monocytes in response to infectious stimuli greatly improves PerC effector activity.

A novel pathway for inducible nitric-oxide synthase activation through inflammasomes.

Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1ß/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1ß and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.

Two physically, functionally, and developmentally distinct peritoneal macrophage subsets.

The peritoneal cavity (PerC) is a unique compartment within which a variety of immune cells reside, and from which macrophages (MØ) are commonly drawn for functional studies. Here we define two MØ subsets that coexist in PerC in adult mice. One, provisionally called the large peritoneal MØ (LPM), contains approximately 90% of the PerC MØ in unstimulated animals but disappears rapidly from PerC following lipopolysaccharide (LPS) or thioglycolate stimulation. These cells express high levels of the canonical MØ surface markers, CD11b and F4/80. The second subset, referred to as small peritoneal MØ (SPM), expresses substantially lower levels of CD11b and F4/80 but expresses high levels of MHC-II, which is not expressed on LPM. SPM, which predominates in PerC after LPS or thioglycolate stimulation, does not derive from LPM. Instead, it derives from blood monocytes that rapidly enter the PerC after stimulation and differentiate to mature SPM within 2 to 4 d. Both subsets show clear phagocytic activity and both produce nitric oxide (NO) in response to LPS stimulation in vivo. However, their responses to LPS show key differences: in vitro, LPS stimulates LPM, but not SPM, to produce NO; in vivo, LPS stimulates both subsets to produce NO, albeit with different response patterns. These findings extend current models of MØ heterogeneity and shed new light on PerC MØ diversity, development, and function. Thus, they introduce a new context for interpreting (and reinterpreting) data from ex vivo studies with PerC MØ.