Alveolar macrophages are epigenetically altered after inflammation, leading to long-term lung immunoparalysis.

Sepsis and trauma cause inflammation and elevated susceptibility to hospital-acquired pneumonia. As phagocytosis by macrophages plays a critical role in the control of bacteria, we investigated the phagocytic activity of macrophages after resolution of inflammation. After resolution of primary pneumonia, murine alveolar macrophages (AMs) exhibited poor phagocytic capacity for several weeks. These paralyzed AMs developed from resident AMs that underwent an epigenetic program of tolerogenic training. Such adaptation was not induced by direct encounter of the pathogen but by secondary immunosuppressive signals established locally upon resolution of primary infection. Signal-regulatory protein α (SIRPα) played a critical role in the establishment of the microenvironment that induced tolerogenic training. In humans with systemic inflammation, AMs and also circulating monocytes still displayed alterations consistent with reprogramming six months after resolution of inflammation. Antibody blockade of SIRPα restored phagocytosis in monocytes of critically ill patients in vitro, which suggests a potential strategy to prevent hospital-acquired pneumonia.

Author Correction: Alveolar macrophages are epigenetically altered after inflammation, leading to long-term lung immunoparalysis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Foamy macrophages and the progression of the human tuberculosis granuloma.

The progression of tuberculosis from a latent, subclinical infection to active disease that culminates in the transmission of infectious bacilli is determined locally at the level of the granuloma. This progression takes place even in the face of a robust immune response that, although it contains infection, is unable to eliminate the bacterium. The factors or environmental conditions that influence this progression remain to be determined. Recent advances have indicated that pathogen-induced dysregulation of host lipid synthesis and sequestration serves a critical role in this transition. The foamy macrophage seems to be a key participant in both sustaining persistent bacteria and contributing to the tissue pathology that leads to cavitation and the release of infectious bacilli.

Expression of CCR6 and CXCR6 by Gut-Derived CD4+/CD8α+ T-Regulatory Cells, Which Are Decreased in Blood Samples From Patients With Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Faecalibacterium prausnitzii, a member of the Clostridium IV group of the Firmicutes phylum that is abundant in the intestinal microbiota, has anti-inflammatory effects. The relative level of F prausnitzii is decreased in fecal samples from patients with inflammatory bowel diseases (IBDs) compared with healthy individuals. Reduced F prausnitzii was correlated with relapse of Crohn's disease after surgery. We identified, in human colonic mucosa and blood, a population of T regulatory type 1-like T regulatory (TREG) cells that express CD4 and CD8α (DP8α T cells) and are specific for F prausnitzii. We aimed to determine whether they are altered in patients with IBD. METHODS: We isolated DP8α T cells from human colon lamina propria and blood samples and used flow cytometry to detect markers of cells that are of colon origin. We quantified DP8α cells that express colon-specific markers in blood samples from 106 patients with IBD, 12 patients with infectious colitis, and 35 healthy donors (controls). We identified cells that respond to F prausnitzii. Cells were stimulated with anti-CD3, and their production of interleukin 10 was measured by enzyme-linked immunosorbent assay. We compared the frequency and reactivity of cells from patients vs controls using the 2-sided Student t test or 1-way analysis of variance. RESULTS: Circulating DP8α T cells that proliferate in response to F prausnitzii express the C-C motif chemokine receptor 6 (CCR6) and C-X-C motif chemokine receptor 6 (CXCR6). These cells also have features of TREG cells, including production of IL-10 and inhibition of T-cell proliferation via CD39 activity. The proportion of circulating CCR6+/CXCR6+ DP8α T cells was significantly reduced (P < .0001) within the total population of CD3+ T cells from patients with IBD compared with patients with infectious colitis or controls. A threshold of <7.875 CCR6+/CXCR6+ DP8α T cells/10,000 CD3+ cells discriminated patients with IBD from those with infectious colitis with 100% specificity and 72.2% sensitivity. CONCLUSIONS: We identified a population of gut-derived TREG cells that are reduced in blood samples from patients with IBD compared with patients with infectious colitis or controls. These cells should be studied further to determine the mechanisms of this reduction and how it might contribute to the pathogenesis of IBD and their prognostic or diagnostic value.

Immunotherapy With Antiprogrammed Cell Death 1 Antibody Improves Outcome in a Mouse Model of Spinal Cord Injury Followed by Staphylococcus aureus Pneumonia.

OBJECTIVES: In patients with spinal cord injury, spinal cord injury-immune depression syndrome induces pneumonia. We aimed to develop a new spinal cord injury-immune depression syndrome mouse model and to test antiprogrammed cell death 1 therapy. DESIGN: Experimental study. SETTING: Research laboratory. SUBJECTS: RjOrl: SWISS and BALB/cJ mice. INTERVENTIONS: Mouse model of spinal cord injury-immune depression syndrome followed by a methicillin-susceptible Staphylococcus aureus pneumonia. Lung injuries were assessed by histologic analysis. Membrane markers and intracytoplasmic cytokines were assessed by flow cytometry. Cytokine production was assessed by quantitative polymerase chain reaction (messenger RNA) and enzyme-linked immunosorbent assay (protein). Animals were treated with blocking antiprogrammed cell death 1 antibodies (intraperitoneal injection). MEASUREMENTS AND MAIN RESULTS: Spinal cord injury mice were more susceptible to methicillin-susceptible S. aureus pneumonia (increased mortality rate). An early inflammatory response was observed in spinal cord injury mice characterized in lungs by a decreased percentage of aerated tissue, an increased production of proinflammatory cytokines (tumor necrosis factor-α). In spleen, an increased expression of major histocompatibility complex class II molecules on dendritic cells, and an increased production of proinflammatory cytokines (interleukin-12, interferon-γ) was observed. Following this pulmonary and systemic inflammation, spinal cord injury-immune depression syndrome was observed in spleens as acknowledged by a decrease of spleen's weight, a lymphopenia, a decrease of major histocompatibility complex class II expression on dendritic cells. An increase of interleukin-10 production and the increase of a cell exhaustion marker expression, programmed cell death 1 receptor on T-cell were also observed. Blockade of programmed cell death 1 molecules, improved survival of spinal cord injury infected mice and enhanced interferon-γ production by natural killer T cells as well as number of viable CD4 T cells. CONCLUSIONS: This model of spinal cord injury in mice mimics a clinical scenario rendering animals prone to a secondary pneumonia. We show for the first time an acute T-cell exhaustion-like phenomenon following an initial inflammatory response. Finally, inhibition of exhaustion pathway should be considered as a new therapeutic option to overcome spinal cord injury-immune depression syndrome and to decrease the rate of nosocomial pneumonia.

FVB/N Mice Spontaneously Heal Ulcerative Lesions Induced by Mycobacterium ulcerans and Switch M. ulcerans into a Low Mycolactone Producer.

Buruli ulcer, a debilitating disease, is caused by Mycobacterium ulcerans. The incidence of this neglected tropical disease is steadily increasing. As a rule, without treatment, skin ulcers occur and a lengthy healing process may be observed associated with severe functional disabilities. Mouse models are already available to study establishment of lesions or evaluation of therapy but a lack of a suitable animal model, mimicking all clinical stages, in particular the healing process, remains an obstacle to understand the pathophysiology of M. ulcerans infection. M. ulcerans was s.c. inoculated in three consanguine mouse strains, that is, BALB/c and C57BL/6, classically used to study mycobacterial infection, and FVB/N. Strikingly, FVB/N mice, although as sensitive as all other mouse strains with respect to M. ulcerans infection, presented a spontaneous healing after the ulcerative phase despite stable bacterial load, and mycolactone toxin was not detected in the healed tissues. The spontaneous healing process was accompanied by an activation of the innate immune system. The adaptive response initiated by FVB/N mice was not involved in the healing process and did not confer protection against M. ulcerans. Our work highlights the importance of innate immune responses to control M. ulcerans infection. This in vivo model of M. ulcerans infection now paves the way for new avenues of research toward the elucidation of critical stages of this disease, such as the characterization of the regulation of mycolactone production, a better understanding of the pathophysiology of M. ulcerans infection, and the development of new therapeutic strategies.

CD4CD8αα lymphocytes, a novel human regulatory T cell subset induced by colonic bacteria and deficient in patients with inflammatory bowel disease.

How the microbiota affects health and disease is a crucial question. In mice, gut Clostridium bacteria are potent inducers of colonic interleukin (IL)-10-producing Foxp3 regulatory T cells (Treg), which play key roles in the prevention of colitis and in systemic immunity. In humans, although gut microbiota dysbiosis is associated with immune disorders, the underlying mechanism remains unknown. In contrast with mice, the contribution of Foxp3 Treg in colitis prevention has been questioned, suggesting that other compensatory regulatory cells or mechanisms may exist. Here we addressed the regulatory role of the CD4CD8 T cells whose presence had been reported in the intestinal mucosa and blood. Using colonic lamina propria lymphocytes (LPL) and peripheral blood lymphocytes (PBL) from healthy individuals, and those with colon cancer and irritable bowel disease (IBD), we demonstrated that CD4CD8αα (DP8α) T lymphocytes expressed most of the regulatory markers and functions of Foxp3 Treg and secreted IL-10. Strikingly, DP8α LPL and PBL exhibited a highly skewed repertoire toward the recognition of Faecalibacterium prausnitzii, a major Clostridium species of the human gut microbiota, which is decreased in patients with IBD. Furthermore, the frequencies of DP8α PBL and colonic LPL were lower in patients with IBD than in healthy donors and in the healthy mucosa of patients with colon cancer, respectively. Moreover, PBL and LPL from most patients with active IBD failed to respond to F. prausnitzii in contrast to PBL and LPL from patients in remission and/or healthy donors. These data (i) uncover a Clostridium-specific IL-10-secreting Treg subset present in the human colonic LP and blood, (ii) identify F. prausnitzii as a major inducer of these Treg, (iii) argue that these cells contribute to the control or prevention of colitis, opening new diagnostic and therapeutic strategies for IBD, and (iv) provide new tools to address the systemic impact of both these Treg and the intestinal microbiota on the human immune homeostasis.

Immune discrepancies during in vitro granuloma formation in response to Cutibacterium (formerly Propionibacterium) acnes infection.

Cutibacterium (formerly Propionibacterium) acnes is involved in chronic/low-grade pathologies such as sarcoidosis or prosthetic joint infection (PJI). In these diseases, granulomatous structures are frequently observed. In this study, we induced a physiological granulomatous reaction in response to different well-characterized clinical C. acnes isolates in order to investigate the cellular process during granuloma formation. Three C. acnes isolates selected according to their origin (PJI, sarcoidosis and acne) were typed by MLST. All C. acnes isolates generated granulomatous structures in our experimental conditions. The bacterial burden was better controlled by granulomas induced by the sarcoidosis C. acnes isolate. The PJI C. acnes isolate, belonging to CC36, promoted the recruitment of CD8+ lymphocytes inside the granuloma. In contrast, the acne and sarcoidosis C. acnes isolates, belonging to phylotypes IA1/CC18 and IA2/CC28, respectively, generated a higher number of granulomas and promoted the recruitment of CD4+ lymphocytes inside the granuloma. Our results provide new evidence supporting the role of C. acnes in the development of sarcoidosis and new explanations concerning the mechanisms underlying PJI due to C. acnes.

High-content screening technology combined with a human granuloma model as a new approach to evaluate the activities of drugs against Mycobacterium tuberculosis.

Tuberculosis remains a major health problem due to the emergence of drug-resistant strains of Mycobacterium tuberculosis. Some models have provided valuable information about drug resistance and efficacy; however, the translation of these results into effective human treatments has mostly proven unsuccessful. In this study, we adapted high-content screening (HCS) technology to investigate the activities of antitubercular compounds in the context of an in vitro granuloma model. We observed significant shifts in the MIC50s between the activities of the compounds under extracellular and granuloma conditions.

Linezolid dampens neutrophil-mediated inflammation in methicillin-resistant Staphylococcus aureus-induced pneumonia and protects the lung of associated damages.

BACKGROUND: Linezolid is considered as a therapeutic alternative to the use of glycopeptides for the treatment of pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA). Clinical studies reported a potent survival advantage conferred by the oxazolidinone and called into question the use of glycopeptides as first-line therapy. METHODS: In a mouse model of MRSA-induced pneumonia, quantitative bacteriology, proinflammatory cytokine concentrations in lung, myeloperoxidase activity, Ly6G immunohistochemistry, and endothelial permeability were assessed to compare therapeutic efficacy and immunomodulative properties of linezolid and vancomycin administered subcutaneously every 12 hours. RESULTS: Significant antibacterial activity was achieved after 48 hours of treatment for linezolid and vancomycin. Levels of interleukin 1ß, a major proinflammatory cytokine, and macrophage inflammatory protein 2, a chemokine involved in the recruitment of neutrophils, were decreased by both antimicrobials. Only linezolid was able to dramatically reduce the production of tumor necrosis factor α. Analysis of myeloperoxidase activity and Ly6G immunostaining showed a dramatic decrease of neutrophil infiltration in infected lung tissues for linezolid-treated animals. A time-dependent increase of endothelial permeability was observed for the control and vancomycin regimens. Of interest, in the linezolid group, decreased endothelial permeability was detected 48 hours after infection. CONCLUSIONS: Our results indicate that linezolid could be superior to vancomycin for the management of MRSA pneumonia by attenuating an excessive inflammatory reaction and protecting the lung from pathogen-associated damages.

Hydrocortisone prevents immunosuppression by interleukin-10+ natural killer cells after trauma-hemorrhage.

OBJECTIVE: Trauma induces a state of immunosuppression, which is responsible for the development of nosocomial infections. Hydrocortisone reduces the rate of pneumonia in patients with trauma. Because alterations of dendritic cells and natural killer cells play a central role in trauma-induced immunosuppression, we investigated whether hydrocortisone modulates the dendritic cell/natural killer cell cross talk in the context of posttraumatic pneumonia. DESIGN: Experimental study. SETTINGS: Research laboratory from an university hospital. SUBJECTS: Bagg Albino/cJ mice (weight, 20-24 g). INTERVENTIONS: First, in an a priori substudy of a multicenter, randomized, double-blind, placebo-controlled trial of hydrocortisone (200 mg/d for 7 d) in patients with severe trauma, we have measured the blood levels of five cytokines (tumor necrosis factor-α, interleukin-6, interleukin-10, interleukin-12, interleukin-17) at day 1 and day 8. In a second step, the effects of hydrocortisone on dendritic cell/natural killer cell cross talk were studied in a mouse model of posttraumatic pneumonia. Hydrocortisone (0.6 mg/mice i.p.) was administered immediately after hemorrhage. Twenty-four hours later, the mice were challenged with Staphylococcus aureus (7 × 10 colony-forming units). MEASUREMENTS AND MAIN RESULTS: Using sera collected during a multicenter study in patients with trauma, we found that hydrocortisone decreased the blood level of interleukin-10, a cytokine centrally involved in the regulation of dendritic cell/natural killer cell cluster. In a mouse model of trauma-hemorrhage-induced immunosuppression, splenic natural killer cells induced an interleukin-10-dependent elimination of splenic dendritic cell. Hydrocortisone treatment reduced this suppressive function of natural killer cells and increased survival of mice with posthemorrhage pneumonia. The reduction of the interleukin-10 level in natural killer cells by hydrocortisone was partially dependent on the up-regulation of glucocorticoid-induced tumor necrosis factor receptor-ligand (TNFsf18) on dendritic cell. CONCLUSIONS: These data demonstrate that trauma-induced immunosuppression is characterized by an interleukin-10-dependent elimination of dendritic cell by natural killer cells and that hydrocortisone improves outcome by limiting this immunosuppressive feedback loop.

Toll-like receptor-4 agonist in post-haemorrhage pneumonia: role of dendritic and natural killer cells.

Haemorrhage-induced immunosuppression has been linked to nosocomial infections. We assessed the impact of monophosphoryl lipid A, a Toll/interleukin-1 receptor-domain-containing adaptor protein inducing interferon-biased Toll-like receptor-4 agonist currently used as a vaccine adjuvant in humans, on post-haemorrhage susceptibility to infection. We used a mouse model of post-haemorrhage pneumonia induced by methicillin-susceptible Staphylococcus aureus. Monophosphoryl lipid A was administered intravenously after haemorrhage and before pneumonia onset. Haemorrhage altered survival rate, increased lung damage (neutrophil accumulation, oedema and cytokine release) and altered the functions of dendritic and natural killer cells. Here, we show that monophosphoryl lipid A decreased systemic dissemination of S. aureus and dampened inflammatory lung lesions. Monophosphoryl lipid A partially restored the capacity for antigen presentation and the transcriptional activity in dendritic cells. Monophosphoryl lipid A did not restore the interferon-γ mRNA but prevented interleukin-10 mRNA overexpression in natural killer cells compared with untreated mice. Ex vivo monophosphoryl lipid A-stimulated dendritic cells or natural killer cells harvested from haemorrhaged animals were adoptively transferred into mice undergoing post-haemorrhage pneumonia. Stimulated dendritic cells (but not stimulated natural killer cells) improved the survival rate compared with mice left untreated. In vivo depletion of natural killer cells decreased survival rate of monophosphoryl lipid A-treated mice. Dendritic and natural killer cells are critically involved in the beneficial effects of monophosphoryl lipid A within post-haemorrhage pneumonia.

An in vitro model of mycobacterial granuloma to investigate the immune response in brain-injured patients.

OBJECTIVE: We investigated the overall immune response to pathogens in brain-injured patients, and assessed its relationship to nosocomial pneumonia. DESIGN: Observational study. SETTING: Two surgical ICUs of a single institution. PATIENTS: Severe brain-injured patients (n = 32) requiring mechanical ventilation and sex- and age-matched healthy donors (n = 25). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We evaluated, ex vivo, the ability of peripheral blood mononuclear cells from brain injury patients to develop an effective granulomatous response to mycobacteria. Thirty-two consecutive patients (25 traumatic brain injured and seven subarachnoid hemorrhage) were included. Median Glasgow Coma Scale was 7 (5-8). Thirteen (41%) patients developed nosocomial pneumonia. Peripheral blood mononuclear cells from brain-injured patients with nosocomial pneumonia generated significantly fewer mature granulomas compared with brain-injured patients without nosocomial pneumonia and with healthy donors. The percentage of multinucleated giant cells was lower in brain-injured patients without nosocomial pneumonia (1% [range: 0%-7%]) and in brain-injured patients with nosocomial pneumonia (4% [range: 2%-5%]) compared with healthy donors (20% [range: 15%-28%]). The blood levels of γδ T cells were significantly increased in brain-injured patients without nosocomial pneumonia (66% [range: 34%-69%]) compared with healthy donors (23% [range: 8%-61%]) and was not altered in brain-injured patients with nosocomial pneumonia (31% [range: 12%-44%]). The percentage of γδ T cells in granulomas was significantly decreased in brain injury patients with nosocomial pneumonia (5% [range: 4%-43%]) compared with healthy donors (43% [range: 19%-54%]) and was not significantly altered in brain-injured patients without nosocomial pneumonia (26% [range: 10%-41%]). The blood levels of natural killer cells were not altered in brain-injured patients. The percentage of natural killer cells in granulomas was significantly decreased in brain-injured patients with nosocomial pneumonia (3% [range: 1%-9%]) compared with brain-injured patients without nosocomial pneumonia (16% [range: 6%-29%]) and with healthy donors (17% [range: 10%-29%]). CONCLUSIONS: Brain-injured patients experienced a maturation defect of the ex vivo granulomatous response involving monocytes as well as natural killer cells and γδ T cells.

The tuberculous granuloma: an unsuccessful host defence mechanism providing a safety shelter for the bacteria?

One of the main features of the immune response to M. Tuberculosis is the formation of an organized structure called granuloma. It consists mainly in the recruitment at the infectious stage of macrophages, highly differentiated cells such as multinucleated giant cells, epithelioid cells and Foamy cells, all these cells being surrounded by a rim of lymphocytes. Although in the first instance the granuloma acts to constrain the infection, some bacilli can actually survive inside these structures for a long time in a dormant state. For some reasons, which are still unclear, the bacilli will reactivate in 10% of the latently infected individuals, escape the granuloma and spread throughout the body, thus giving rise to clinical disease, and are finally disseminated throughout the environment. In this review we examine the process leading to the formation of the granulomatous structures and the different cell types that have been shown to be part of this inflammatory reaction. We also discuss the different in vivo and in vitro models available to study this fascinating immune structure.

Human gut microbiota-reactive DP8α regulatory T cells, signature and related emerging functions.

In mice, microbiota-induced Tregs both maintain intestinal homeostasis and provide resistance to immuno-pathologies in the adult. Identifying their human functional counterpart therefore represents an important goal. We discovered, in the human colonic lamina propria and blood, a FoxP3-negative IL-10-secreting Treg subset, which co-expresses CD4 and CD8α (hence named DP8α) and displays a TCR-reactivity against Faecalibacterium prausnitzii, indicating a role for this symbiotic bacterium in their induction. Moreover, supporting their role in intestinal homeostasis, we previously reported both their drastic decrease in IBD patients and their protective role in vivo against intestinal inflammation, in mice. Here, we aimed at identifying the genomic, phenotypic and functional signatures of these microbiota-induced Tregs, towards delineating their physiological role(s) and clinical potential. Human F. prausnitzii-reactive DP8α Treg clones were derived from both the colonic lamina propria and blood. RNA-sequencing, flow cytometry and functional assays were performed to characterize their response upon activation and compare them to donor- and tissue-matched FoxP3+ Treg clones. DP8α Tregs exhibited a unique mixed Tr1-like/cytotoxic CD4+ T cell-profile and shared the RORγt and MAF master genes with mouse gut microbiota-induced FoxP3+ Tregs. We revealed their potent cytotoxic, chemotactic and IgA-promoting abilities, which were confirmed using in vitro assays. Therefore, besides their induction by a Clostridium bacterium, DP8α Tregs also partake master genes with mouse microbiota-induced Tregs. The present identification of their complete signature and novel functional properties, should be key in delineating the in vivo roles and therapeutic applications of these unique human microbiota-induced Tregs through their study in pathological contexts, particularly in inflammatory bowel diseases.

Human CD4+CD8α+ Tregs induced by Faecalibacterium prausnitzii protect against intestinal inflammation.

Abundance of Faecalibacterium prausnitzii, a dominant bacterium of the human microbiota that exhibits antiinflammatory effects, is decreased in patients with inflammatory bowel diseases (IBD). In humans, colonic lamina propria contains IL-10-secreting, Foxp3- Tregs characterized by a double expression of CD4 and CD8α (DP8α) and a specificity for F. prausnitzii. This Treg subset is decreased in IBD. The in vivo effect of DP8α cells has not been evaluated yet to our knowledge. Here, using a humanized model of a NSG immunodeficient mouse strain that expresses the HLA D-related allele HLA-DR*0401 but not murine class II (NSG-Ab° DR4) molecules, we demonstrated a protective effect of a HLA-DR*0401-restricted DP8α Treg clone combined with F. prausnitzii administration in a colitis model. In a cohort of patients with IBD, we showed an independent association between the frequency of circulating DP8α cells and disease activity. Finally, we pointed out a positive correlation between F. prausnitzii-specific DP8α Tregs and the amount of F. prausnitzii in fecal microbiota in healthy individuals and patients with ileal Crohn's disease.

Comparison of the moonlighting actions of the two highly homologous chaperonin 60 proteins of Mycobacterium tuberculosis.

Evidence is emerging that the two chaperonin (Cpn) 60 proteins of Mycobacterium tuberculosis, Cpn60.1 and Cpn60.2, have moonlighting actions that may contribute to the pathology of tuberculosis. We studied the release of Cpn60.1 from M. tuberculosis and infected macrophage like cells and compared recombinant Cpn60.1 and Cpn60.2 in a range of cell-based assays to determine how similar the actions of these highly homologous proteins are. We now establish that Cpns are similar as follows: (i) Cpn60.1, as it has been shown for Cpn60.2, is released by M. tuberculosis in culture, and Cpn60.1 is furthermore released when the bacterium is in quiescent, but not activated, macrophage like cells, and (ii) both proteins only showed a partial requirement for MyD88 for the induction of proinflammatory cytokine production compared to lipopolysaccharide. However, we also found major differences in the cellular action of Cpns. (i) Cpn60.2 proved to be a more potent stimulator of whole blood leukocytes than Cpn60.1 and was the only one to induce tumor necrosis factor alpha synthesis. (ii) Cpn60.1 bound to ca. 90% of circulating monocytes compared to Cpn60.2, which bound <50% of these cells. Both chaperonins bound to different cell surface receptors, while monocyte activation by both proteins was completely abrogated in TLR4-/- mice, although Cpn60.2 also showed significant requirement for TLR2. Finally, an isogenic mutant lacking cpn60.1, but containing intact cpn60.2, was severely inhibited in generating multinucleate giant cells in an in vitro human granuloma assay. These results clearly show that, despite significant sequence homology, M. tuberculosis Cpn60 proteins interact in distinct ways with human or murine macrophages.

Low penetrance, broad resistance, and favorable outcome of interleukin 12 receptor beta1 deficiency: medical and immunological implications.

The clinical phenotype of interleukin 12 receptor beta1 chain (IL-12Rbeta1) deficiency and the function of human IL-12 in host defense remain largely unknown, due to the small number of patients reported. We now report 41 patients with complete IL-12Rbeta1 deficiency from 17 countries. The only opportunistic infections observed, in 34 patients, were of childhood onset and caused by weakly virulent Salmonella or Mycobacteria (Bacille Calmette-Guérin -BCG- and environmental Mycobacteria). Three patients had clinical tuberculosis, one of whom also had salmonellosis. Unlike salmonellosis, mycobacterial infections did not recur. BCG inoculation and BCG disease were both effective against subsequent environmental mycobacteriosis, but not against salmonellosis. Excluding the probands, seven of the 12 affected siblings have remained free of case-definition opportunistic infection. Finally, only five deaths occurred in childhood, and the remaining 36 patients are alive and well. Thus, a diagnosis of IL-12Rbeta1 deficiency should be considered in children with opportunistic mycobacteriosis or salmonellosis; healthy siblings of probands and selected cases of tuberculosis should also be investigated. The overall prognosis is good due to broad resistance to infection and the low penetrance and favorable outcome of infections. Unexpectedly, human IL-12 is redundant in protective immunity against most microorganisms other than Mycobacteria and Salmonella. Moreover, IL-12 is redundant for primary immunity to Mycobacteria and Salmonella in many individuals and for secondary immunity to Mycobacteria but not to Salmonella in most.

Foamy macrophages from tuberculous patients' granulomas constitute a nutrient-rich reservoir for M. tuberculosis persistence.

Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis-infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria.

FoxP3+ regulatory T cells suppress early stages of granuloma formation but have little impact on sarcoidosis lesions.

Sarcoidosis is characterized by a disproportionate Th1 granulomatous immune response in involved organs. It is also associated with both peripheral and intratissular regulatory T cell (Treg) expansion. These cells exhibit powerful antiproliferative activity, yet do not completely inhibit the production of either tumor necrosis factor-alpha or interferon-gamma. The origin of the observed Treg amplification and, more importantly, its impact on the evolution of sarcoidosis remain unresolved issues. Here, we show that CD4(+)CD45RA(-)FoxP3(bright) Tregs proliferate and accumulate within granulomas. However, circulating and tissue Treg numbers are neither correlated with the dissemination of the disease nor correlated locally with the extent of granulomatous inflammation. Rather, we found a positive correlation between the presence of Tregs in renal granulomas and the degree of interstitial fibrosis (r = 0.46, P = 0.03, n = 20). Furthermore, Treg depletion accelerates in vitro granuloma growth in mononuclear cell cultures of healthy controls, but not in those from patients with active sarcoidosis. The results of this study show that although healthy Tregs suppress the initial steps of granuloma formation, they have no positive influence on sarcoidosis lesions. Our findings argue for a more preventive than curative effect of Tregs on inflammatory processes.