NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis.

BACKGROUND:: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1β and IL-18. METHODS:: In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3(-/-) and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6-trinitrobenzenesulfonic acid models of experimental colitis. RESULTS:: Nlrp3(-/-) mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL-1β, reduced antiinflammatory cytokine IL-10, and reduced protective growth factor TGF-β. Macrophages isolated from Nlrp3(-/-) mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3-deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3(-/-) mice displayed altered colonic β-defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota. CONCLUSIONS:: Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations. (Inflamm Bowel Dis 2010).

Gas6 and its receptors are implicated in sepsis as modulators of innate immunity

Gas6 downregulates the activation state of macrophages and thereby their production of proinflammatory cytokines induced by various stimuli. We aimed to determine whether Gas6 is involved in sepsis. We measured Gas6 plasma levels in 13 healthy subjects, 29 patients with severe sepsis, and 18 patients with non-infectious inflammatory diseases. Gas6 level was higher in septic patients than in control groups (P 0.0001). The sensitivity and specificity of Gas6 levels to predict fatal outcome were 83% and 88%. We next investigated whether Gas6 affects cytokine production and outcome in experimental models of endotoxemia and peritonitis in wild-type (WT) and Gas6-/- mice. Circulating levels of Gas6 after LPS 25mg/kg i.p. peaked at 1 hour (P<0.001). Similarly, TNF- was higher in Gas6-/- than in WT mice 1 hour after LPS (P<0.05). Furthermore, 62 anti- and pro-inflammatory cytokines were quantified in plasma after LPS injection. Their levels were globally higher in Gas6-/- plasma after LPS, 47/62 cytokines being at least 50% higher in Gas6-/- than in WT plasma after 1 hour. Mortality induced by 25mg/kg LPS was 25% in WT versus 87% in Gas6-/- mice (P<0.05). LPS-induced mortality in Gas6 receptors Axl-/-, Tyro3-/- and Merkd was also enhanced when compared to WT mice (P<0.001). In peritonitis models (cecal ligation and puncture, CLP, and i.p. injection of E. coli), Gas6 plasma levels increased and remained elevated at least 24 hours. CLP increased mortality in Gas6-/- mice. Finally, we explored the role of Gas6 in LPS-treated macrophages. We found that Gas6 was released by LPS-stimulated WT macrophages and that Gas6-/- macrophages produced more TNF- and IL-6 than WT macrophages. Cytokine release by Gas6-/- macrophages was higher than by WT macrophages (cytokine array). Adjunction of recombinant Gas6 to the culture medium of Gas6-/- macrophages diminished the cytokine production to WT levels. In LPS-treated Gas6-/- macrophages, Akt and Erk1/2 phosphorylation was reduced whereas p38 and NF B activation was enhanced. Thus, in septic patients, elevated Gas6 levels were associated with fatal outcome. In mice, they raised in experimental endotoxemia and peritonitis models, and correlated also with sepsis severity. However, Gas6-/- mice survival in these models was reduced compared to WT. Gas6 secreted by macrophages in response to LPS activated Akt and restrained p38 and NF B activation, thereby dampening macrophage activation. Altogether these data suggest that, during endotoxemia, Gas6-/- mice phenotype resembles that of mice which have undergone PI3K inhibition, indicating that Gas6 is a major modulator of innate immunity.

Distinct ultrastructural aspects in different biopsies of a single patient with diffuse cutaneous leishmaniasis

The authors investigated the relation between parasites and host-cells in active and regressed lesions of a patient with diffuse cutaneous leishmaniasis, evaluating the frequency of different cell types, and the location and integrity of amastigotes. No correlation was found between parasite integrity and size of parasitophorous vacuoles. They observed ultrastructural findings characterizing a cell mediated immune response: macrophages lysis, parasitic destruction inside macrophages, close contact between parasitized macrophages and lymphocytes and between parasites and lymphocytes, lymphocytic infiltration and fibrosis. They suggest that in DCL there is a limited cellular immune response, although insufficient to control infection.

Trypanosoma cruzi: experimental Chagas' disease in Rhesus monkeys. II. Ultraestructural and cytochemical studies of peroxidase and acid phosphatase activities

Ultrastructural and cytochemical studies of peroxidase and acid phosphatase were performed in skin, lymph node and heart muscle tissue of thesus monkeys with experimental Chagas's disease. At the site of inoculation ther was a proliferative reaction with the presence of immature macrophages revealed by peroxidase technique. At the lymph node a difuse inflammatory exudate with mononuclear cells, fibroblasts and immature activated macrophages reproduces the human patrtern of acute Chagas' disease inflamatory lesions. The hearth muscle cells present different degrees of degenerative alterations and a striking increase in the number of lysosomal profiles that exhibit acid hydrolase reaction product. A strong inflammatory reaction was present due to lymphocytic infiltrate or due to eosinophil granulocytes associated to ruptured cells. The present study provides some experimental evidences that the monkey model could be used as a reliable model to characterize histopathological alterations of the human disease.

Reversibility of cardiac fibrosis in mice chronically infected with Trypanosoma cruzi, under specific chemotherapy

This investigation was performed to verify the effect of specific chemotherapy (Benznidazole or MK-346) on the inflammatory and fibrotic cardiac alterations in mice chronically infected with the strains 21 SF (Type II) and Colombian (Type III) of Trypanosoma cruzi. To obtain chronically infected mice, two groups of 100 Swiss mice each, were infected with either the 21 SF or the Colombian strain (2x 10 [raised to the power of] 4 and 5x 10 [raised to the power of] 4 blood forms respectively). The rate of morality in the acute phase was of 80% for both groups. Twenty surviving mice chronically infected with the 21 SF strain and 20 with the Colombian strain were then divided in treated and untreated groups. Excluding those that died during the course of treatment, 14 mice chronically infected with the 21 SF strain and 15 with the Colombian strain were evaluated in the present study. Chemotherapy was performed with Benznidazole (N-benzil-2-nitro-1-imidazolacetamide) in the dose of 100mg/k.b.w/day, for 60 days, or with the MK-436(3(1-methyl-5 nitroimidazol-2-yl) in two daily doses of 250 mg/k.b.w, for 20 days. Parasitological cure tests were performed (xenodiagnosis, haemoculture, subinovulation of the blood into newborn mice), and serological indirect immunofluorescence test. The treated and untreated mice as well as intact controls were killed at different periods after treatment and the heart were submitted to histopathological study with hematoxilineosin and picrosirius staining; ultrastructural study; collagen immunotyping, fibronectin and laminin identification by immunofluorescence tests. Results: the untreated controls either infected with 21 SF or Colombian strain, showed inflammatory and fibrotic alterations that were mild to moderate with the 21 SF strain and intense with the Colombian strain. Redpicrosirius staining showed bundles of collagen in the interstitial space and around cardiac fibers. Increased deposits of mitritial components and collagen fibers, macrophages and fibroblasts appeared at the ultra structural examination. Deposits of fibronectin, laminin, pro-III and IV collagens were seen, most intense in those infected with the Colombian strain. Treated nice, parasitologically cured, presented clear-cut regression of the inflammatory lesions and of the interstitial matrix thickening. Mice infected with the Colombian strain and treated with MK-436, was parasitologically cured in 5/6 cases and showed mild inflammatory infiltration and fibrosis. The mice treated with Benznidazole (Colombian strain) did not cure and showed moderate fibrosis and inflammation. Treatment of the nice infected with the 21 SF with Benznidazole determined parasitological cure of all animals, that showed mild inflammation and fibrosis of the myocardium. The cured mice of all groups and treated but uncured showed collagen degradation at electronmicroscopy and decrease of immunofluorescence pattern of the matrix.

NALP3 is not necessary for early protection against experimental tuberculosis.

In vitro, Toll-like receptors (TLR)2, 4 and 9 as well as NOD-like receptor 2 critically determine macrophage responses to Mycobacterium tuberculosis (Mtb) infection. However, in low-dose experimental murine tuberculosis, single or multiple deficiencies in TLRs 2, 4, 9 or NOD2 have little, if any, impact on early mycobacterial growth containment, granuloma formation and survival. Here, we analyzed the relevance of NALP3, one component of the danger-signaling inflammasome, for (i) Mtb-induced cytokine secretion in vitro and in vivo, (ii) restriction of Mtb replication in infected organs and (iii) granuloma formation. In the absence of functional NALP3, there was no IL-1beta and IL-18 production in Mtb-infected dendritic cells and macrophages in vitro, whereas secretion of IL-1alpha, IL-12p40 and TNF remained unaffected. After three weeks of infection, NALP3-deficient as well as IL-18-deficient mice were as capable as wildtype mice of restricting Mtb loads at a plateau level within well-differentiated granulomas. In conclusion, despite its involvement in cytokine processing, NALP3 is not essential for induction of protective immunity to Mtb.

The murine model of infection with Leishmania major and its importance for the deciphering of mechanisms underlying differences in Th cell differentiation in mice from different genetic backgrounds.

Mice from the majority of inbred strains are resistant to infection by Leishmania major, an obligate intracellular protozoan parasite of macrophages in the mammalian host. In contrast, mice from BALB strains are unable to control infection and develop progressive disease. In this model of infection, genetically determined resistance and susceptibility have been clearly shown to result from the appearance of parasite-specific CD4+ T helper 1 or T helper 2 cells, respectively. This murine model of infection is considered as one of the best experimental systems for the study of the mechanisms operating in vivo at the initiation of polarised T helper 1 and T helper 2 cell maturation. Among the several factors influencing Th cell development, cytokines themselves critically regulate this process. The results accumulated during the last years have clarified some aspects of the role played by cytokines in Th cell differentiation. They are providing critical information that may ultimately lead to the rational devise of means by which to tailor immune responses to the effector functions that are most efficient in preventing and/or controlling infections with pathogens.

Trypanosoma cruzi: effect of phenothiazines on the parasite and its interaction with host cells

Phenothiazines were observed to have a direct effect on Trypanosoma cruzi and on its in vitro interaction with host cells. They caused lysis of trypomastigotes (50 uM/24 h) and,to a lesser extent, epimastigote proliferation. Treatment of infected peritoneal macrophages with 12.5 uM chlorpromazine or triflupromazine inhibited the infection; this effect was found to be partially reversible if the drugs were removed after 24 h of treatment. At 60 uM, the drugs caused damage to amastigotes interiorized in heart muscle cells. However, the narrow margin of toxity between anti-trypanossomal activity and damage to host cells mitigates against in vivo investigation at the present time. Possible hypothesis for the mechanism of action of phenothiazines are discussed.

Contribution of TIR domain-containing adapter inducing IFN-beta-mediated IL-18 release to LPS-induced liver injury in mice.

BACKGROUND/AIMS: After treatment with heat-killed Propionibacterium acnes mice show dense hepatic granuloma formation. Such mice develop liver injury in an interleukin (IL)-18-dependent manner after challenge with a sublethal dose LPS. As previously shown, LPS-stimulated Kupffer cells secrete IL-18 depending on caspase-1 and Toll-like receptor (TLR)-4 but independently of its signal adaptor myeloid differentiation factor 88 (MyD88), suggesting importance of another signal adaptor TIR domain-containing adapter inducing IFN-beta (TRIF). Nalp3 inflammasome reportedly controls caspase-1 activation. Here we investigated the roles of MyD88 and TRIF in P. acnes-induced hepatic granuloma formation and LPS-induced caspase-1 activation for IL-18 release. METHODS: Mice were sequentially treated with P. acnes and LPS, and their serum IL-18 levels and liver injuries were determined by ELISA and ALT/AST measurement, respectively. Active caspase-1 in LPS-stimulated Kupffer cells was determined by Western blotting. RESULTS: Macrophage-ablated mice lacked P. acnes-induced hepatic granuloma formation and LPS-induced serum IL-18 elevation and liver injury. Myd88(-/-) Kupffer cells, but not Trif(-/-) cells, exhibited normal caspase-1 activation upon TLR4 engagement in vitro. Myd88(-/-) mice failed to develop hepatic granulomas after P. acnes treatment and liver injury induced by LPS challenge. In contrast, Trif(-/-) mice normally formed the hepatic granulomas, but could not release IL-18 or develop the liver injury. Nalp3(-/-) mice showed the same phenotypes of Trif(-/-) mice. CONCLUSIONS: Propionibacterium acnes treatment MyD88-dependently induced hepatic granuloma formation. Subsequent LPS TRIF-dependently activated caspase-1 via Nalp3 inflammasome and induced IL-18 release, eventually leading to the liver injury.

The sirtuin inhibitor cambinol impairs MAPK signaling, inhibits inflammatory and innate immune responses and protects from septic shock.

Sirtuins (SIRT1-7) are NAD(+)-dependent histone deacetylases (HDACs) that play an important role in the control of metabolism and proliferation and the development of age-associated diseases like oncologic, cardiovascular and neurodegenerative diseases. Cambinol was originally described as a compound inhibiting the activity of SIRT1 and SIRT2, with efficient anti-tumor activity in vivo. Here, we studied the effects of cambinol on microbial sensing by mouse and human immune cells and on host innate immune responses in vivo. Cambinol inhibited the expression of cytokines (TNF, IL-1β, IL-6, IL-12p40, and IFN-γ), NO and CD40 by macrophages, dendritic cells, splenocytes and whole blood stimulated with a broad range of microbial and inflammasome stimuli. Sirtinol, an inhibitor of SIRT1 and SIRT2 structurally related to cambinol, also decreased macrophage response to TLR stimulation. On the contrary, selective inhibitors of SIRT1 (EX-527 and CHIC-35) and SIRT2 (AGK2 and AK-7) used alone or in combination had no inhibitory effect, suggesting that cambinol and sirtinol act by targeting more than just SIRT1 and SIRT2. Cambinol and sirtinol at anti-inflammatory concentrations also did not inhibit SIRT6 activity in in vitro assay. At the molecular level, cambinol impaired stimulus-induced phosphorylation of MAPKs and upstream MEKs. Going well along with its powerful anti-inflammatory activity, cambinol reduced TNF blood levels and bacteremia and improved survival in preclinical models of endotoxic shock and septic shock. Altogether, our data suggest that pharmacological inhibitors of sirtuins structurally related to cambinol may be of clinical interest to treat inflammatory diseases.

USP2-45 represses aldosterone mediated responses by decreasing mineralocorticoid receptor availability.

BACKGROUND/AIMS: Ligand activation of the mineralocorticoid receptor (MR) induces several post-translational modifications (PTMs). Among the different PTMs, MR is known to be dynamically ubiquitylated with impact on its stability and transcriptional activity. Previously, we have shown that MR is monoubiquitylated at the basal state and that aldosterone stimulation induces monoubiquitylation removal prompting polyubiquitin-dependent destabilization of the receptor and proteasomal degradation. This study investigated the role of the aldosterone induced ubiquitin-specific protease USP2-45 on the ubiquitylation state of MR. METHODS: Renal epithelial cells M1 were co-transfected with MR with or without wild-type or inactive USP2-45. The association of MR with USP2-45 or TSG101 as well as MR ubiquitylation state were determined by immunoprecipitation and immunoblotting. MR transcriptional activity was assessed via a luciferase reporter gene. RESULTS: We show that USP2-45 is able to bind MR and, similarly to aldosterone, induce MR monoubiquitylation removal, disruption of MR/TSG101 association and destabilization of MR at protein level. CONCLUSION: This study provides a novel role for USP2-45 by playing a pivotal role in the regulation of the ubiquitylation state of MR and reveals the existence of a negative feedback loop for limiting the aldosterone induced response.

Macrophage activation and histopathological findings in Calomys callosus and Swiss mice infected with several strains of Trypanosoma cruzi

Peritoneal macrophage activation as measured by H2O2 release and histopathology was compared between Swiss mice and Calomys callosus, a wild rodent, reservoir of Trypanosoma cruzi, during the course of infection with four strains of this parasite. In mice F and Y strain infections result in high parasitemia and mortality while with silvatic strains Costalimai and M226 parasitemia is sub-patent, with very low mortality. H2O2 release peaked at 33,6 and 59 nM/2 x 10(elevado a sexta potência) cells for strains Y and F, respectively, 48 and 50 nM/2 x 10 (elevado a sexta potência) for strains Costalimai and M226, at different days after infection. Histopathological findings of myositis, myocarditis, necrotizing artheritis and abscence of macrophage parasitism were foud for strains F and Costalimai. Y strain infection presented moderate myocarditis and myositis, with parasites multiplying within macrophages. In C. callosus all four strains resulted in patent parasitemia wich was eventually overcome, with scarce mortality. H2O2 release for strains Y or F was comparable to that of mice-peaks of 27 and 53 nM/2 x 10 (elevado a sexta potência) cells, with lower values for strains Costalimai and M226 - 16.5 and 4.6 nM/2 x 10(elevado a sexta potência)cells, respectively. Histopathological lesions with Y and F strain injected animals were comparable to those of mice at the onset of infections; they subsided completely at the later stages with Y strain and partially with F strain infected C. callosus. In Costalimai infected C. callosus practically no histopathological alterations were observed.

Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

Dengue 2 virus enhancement in asthmatic and non asthmatic individual

During the 1981 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) Cuban epidemic, bronchial asthma (BA) was frequently found as a personal or family antecedent in dengue hemorragic fever patients. Considering that antibody dependent enhancement (ADE) plays an important role in the etiopathogenic mechanism of DHF/DSS, we decide to study the Dengue 2 virus (D2V) capability of replication in peripheral blood leukocytes (PBL) from asthmatic patients and healthy persons. In 90% of asthmatic patients and 53.8% of control group it was obtained PBL with a significant D2V enhancing activity (X² p < 0.01). Power enhancement was higher in asthmatic group. This is the first in vitro study relating BA and the dengue 2 virus immuno enhancement. The results obtained support the role of BA as a risk factor for DHF/DSS as already described on epidemiological data.

Cytokines and dysregulation of the immune response in human malaria

The dysregulation of the immune response by malaria parasite has been considered as a possible constraint to the effectiveness of malaria vaccination. In spite of the important role interleukin-I (IL-1) in malaria are lacking. We found that only 2 out of 35 subjectswith acute malaria showed increased levels of serum IL-1 alpha by enzyme immunoassay. To assess whether IL-1 could interfere with T- lymphocyte responses, blood mononuclear cells from patients infected with Plasmodium falciparum, P. vivax, or healthy subjects were cultured with phytohemagglutinin, and lymphocyte proliferation measured 72h later by 3H-thymidine incorporation. Our data showed that T-lymphocyte responses are depressed both in P. falciparum (10,500 ñ 2,900) and P. vivax malaria (13,000 ñ 3,300), as compared to that of healthy individuals (27,000 ñ 3,000). Addition of IL-1 partially reserved depression of malaria lymphocytes, but had no effect on normal cells. On the other hand, T-lymphocytes from malaria infected-subjects presented a minimal decrease in proliferation, when cultured in the presence of exogenous PGE2. These data indicate the occurrence of two defects of immunoregulation in malaria: a deficiency of IL-1 production by monocytes/macrophages, and an increased resistance of lymphocytes to the antiproliferative effect of PGE2.