Human metapneumovirus respiratory infection affects both innate and adaptive intestinal immunity.

Introduction: Respiratory infections are one of the leading causes of morbidity and mortality worldwide, mainly in children, immunocompromised people, and the elderly. Several respiratory viruses can induce intestinal inflammation and alterations in intestinal microbiota composition. Human metapneumovirus (HMPV) is one of the major respiratory viruses contributing to infant mortality in children under 5 years of age worldwide, and the effect of this infection at the gut level has not been studied. Methods: Here, we evaluated the distal effects of HMPV infection on intestinal microbiota and inflammation in a murine model, analyzing several post-infection times (days 1, 3, and 5). Six to eight-week-old C57BL/6 mice were infected intranasally with HMPV, and mice inoculated with a non-infectious supernatant (Mock) were used as a control group. Results: We did not detect HMPV viral load in the intestine, but we observed significant changes in the transcription of IFN-γ in the colon, analyzed by qPCR, at day 1 post-infection as compared to the control group. Furthermore, we analyzed the frequencies of different innate and adaptive immune cells in the colonic lamina propria, using flow cytometry. The frequency of monocyte populations was altered in the colon of HMPV -infected mice at days 1 and 3, with no significant difference from control mice at day 5 post-infection. Moreover, colonic CD8+ T cells and memory precursor effector CD8+ T cells were significantly increased in HMPV-infected mice at day 5, suggesting that HMPV may also alter intestinal adaptive immunity. Additionally, we did not find alterations in antimicrobial peptide expression, the frequency of colonic IgA+ plasma cells, and levels of fecal IgA. Some minor alterations in the fecal microbiota composition of HMPV -infected mice were detected using 16s rRNA sequencing. However, no significant differences were found in ß-diversity and relative abundance at the genus level. Discussion: To our knowledge, this is the first report describing the alterations in intestinal immunity following respiratory infection with HMPV infection. These effects do not seem to be mediated by direct viral infection in the intestinal tract. Our results indicate that HMPV can affect colonic innate and adaptive immunity but does not significantly alter the microbiota composition, and further research is required to understand the mechanisms inducing these distal effects in the intestine.

Immune Responses at Host Barriers and Their Importance in Systemic Autoimmune Diseases.

Host barriers such as the skin, the lung mucosa, the intestinal mucosa and the oral cavity are crucial at preventing contact with potential threats and are populated by a diverse population of innate and adaptive immune cells. Alterations in antigen recognition driven by genetic and environmental factors can lead to autoimmune systemic diseases such rheumatoid arthritis, systemic lupus erythematosus and food allergy. Here we review how different immune cells residing at epithelial barriers, host-derived signals and environmental signals are involved in the initiation and progression of autoimmune responses in these diseases. We discuss how regulation of innate responses at these barriers and the influence of environmental factors such as the microbiota can affect the susceptibility to develop local and systemic autoimmune responses particularly in the cases of food allergy, systemic lupus erythematosus and rheumatoid arthritis. Induction of pathogenic autoreactive immune responses at host barriers in these diseases can contribute to the initiation and progression of their pathogenesis.

Uptake of Tropheryma whipplei by Intestinal Epithelia.

BACKGROUND: Tropheryma whipplei (TW) can cause different pathologies, e.g., Whipple's disease and transient gastroenteritis. The mechanism by which the bacteria pass the intestinal epithelial barrier, and the mechanism of TW-induced gastroenteritis are currently unknown. METHODS: Using ex vivo disease models comprising human duodenal mucosa exposed to TW in Ussing chambers, various intestinal epithelial cell (IEC) cultures exposed to TW and a macrophage/IEC coculture model served to characterize endocytic uptake mechanisms and barrier function. RESULTS: TW exposed ex vivo to human small intestinal mucosae is capable of autonomously entering IECs, thereby invading the mucosa. Using dominant-negative mutants, TW uptake was shown to be dynamin- and caveolin-dependent but independent of clathrin-mediated endocytosis. Complementary inhibitor experiments suggested a role for the activation of the Ras/Rac1 pathway and actin polymerization. TW-invaded IECs underwent apoptosis, thereby causing an epithelial barrier defect, and were subsequently subject to phagocytosis by macrophages. CONCLUSIONS: TW enters epithelia via an actin-, dynamin-, caveolin-, and Ras-Rac1-dependent endocytosis mechanism and consecutively causes IEC apoptosis primarily in IECs invaded by multiple TW bacteria. This results in a barrier leak. Moreover, we propose that TW-packed IECs can be subject to phagocytic uptake by macrophages, thereby opening a potential entry point of TW into intestinal macrophages.

Intestinal barrier dysfunction mediates Whipple's disease immune reconstitution inflammatory syndrome (IRIS).

BACKGROUND & AIMS: Classical Whipple's disease (CWD) affects the gastrointestinal tract and causes chronic diarrhea, malabsorption, and barrier dysfunction with microbial translocation (MT). Immune reconstitution inflammatory syndrome (IRIS) is a serious complication during antimicrobial treatment of CWD. The pathomechanisms of IRIS have not been identified and mucosal barrier integrity has not been studied in patients with IRIS CWD. METHODS: In 96 CWD patients (n = 23 IRIS, n = 73 non-IRIS) and 30 control subjects, we analysed duodenal morphology by histology, measured serum markers of MT, and proinflammatory cytokines in biopsy supernatants, and correlated microbial translocation with T cell reconstitution and activation. RESULTS: Before treatment, duodenal specimens from patients who later developed IRIS exhibited a more pronounced morphological transformation that suggested a disturbed barrier integrity when compared with the non-IRIS group. Villous atrophy was mediated by increased apoptosis of epithelial cells, which was insufficiently counterbalanced by regenerative proliferation of crypt cells. Pretreatment deficiencies in the mucosal secretion of proinflammatory cytokines and chemokines (e.g., IL-6, CCL2) in these patients markedly resolved after therapy induction. High serum levels of lipopolysaccharides (LPS), soluble CD14 (sCD14), and LPS-binding protein (LBP) combined with low endotoxin core antibody (EndoCAb) titres suggested systemic MT in CWD patients developing IRIS. CD4+ T cell count and activation in IRIS CWD patients correlated positively with sCD14 levels and negatively with EndoCAb titres. Furthermore, the degree of intestinal barrier dysfunction and MT was predictive for the onset of IRIS. CONCLUSION: Prolonged MT across a dysfunctional intestinal mucosal barrier due to severe tissue damage favors dysbalanced immune reconstitution and systemic immune activation in IRIS CWD. Therefore, the monitoring of inflammatory and MT markers in CWD patients might be helpful in identifying patients who are at risk of developing IRIS. Therapeutic strategies to reconstitute the mucosal barrier and control inflammation could assist in the prevention of IRIS.

Regulation of Tolerogenic Features on Dexamethasone-Modulated MPLA-Activated Dendritic Cells by MYC.

The potential of tolerogenic dendritic cells (tolDCs) to shape immune responses and restore tolerance has turn them into a promising therapeutic tool for cellular therapies directed toward immune regulation in autoimmunity. Although the cellular mechanisms by which these cells can exert their regulatory function are well-known, the mechanisms driving their differentiation and function are still poorly known, and the variety of stimuli and protocols applied to differentiate DCs toward a tolerogenic phenotype makes it even more complex to underpin the molecular features involved in their function. Through transcriptional profiling analysis of monocyte-derived tolDCs modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), known as DM-DCs, we were able to identify MYC as one of the transcriptional regulators of several genes differentially expressed on DM-DCs compared to MPLA-matured DCs (M-DCs) and untreated/immature DCs (DCs) as revealed by Ingenuity Pathway Analysis (IPA) upstream regulators evaluation. Additionally, MYC was also amidst the most upregulated genes in DM-DCs, finding that was confirmed at a transcriptional as well as at a protein level. Blockade of transactivation of MYC target genes led to the downregulation of tolerance-related markers IDO1 and JAG1. MYC blockade also led to downregulation of PLZF and STAT3, transcription factors associated with immune regulation and inhibition of DC maturation, further supporting a role of MYC as an upstream regulator contributing to the regulatory phenotype of DM-DCs. On the other hand, we had previously shown that fatty acid oxidation, oxidative metabolism and zinc homeostasis are amongst the main biological functions represented in DM-DCs, and here we show that DM-DCs exhibit higher intracellular expression of ROS and Zinc compared to mature M-DCs and DCs. Taken together, these findings suggest that the regulatory profile of DM-DCs is partly shaped by the effect of the transcriptional regulation of tolerance-inducing genes by MYC and the modulation of oxidative metabolic processes and signaling mediators such as Zinc and ROS.

Humanized Mouse Models of Rheumatoid Arthritis for Studies on Immunopathogenesis and Preclinical Testing of Cell-Based Therapies.

Rodent models of rheumatoid arthritis (RA) have been used over decades to study the immunopathogenesis of the disease and to explore intervention strategies. Nevertheless, mouse models of RA reach their limit when it comes to testing of new therapeutic approaches such as cell-based therapies. Differences between the human and the murine immune system make it difficult to draw reliable conclusions about the success of immunotherapies. To overcome this issue, humanized mouse models have been established that mimic components of the human immune system in mice. Two main strategies have been pursued for humanization: the introduction of human transgenes such as human leukocyte antigen molecules or specific T cell receptors, and the generation of mouse/human chimera by transferring human cells or tissues into immunodeficient mice. Recently, both approaches have been combined to achieve more sophisticated humanized models of autoimmune diseases. This review discusses limitations of conventional mouse models of RA-like disease and provides a closer look into studies in humanized mice exploring their usefulness and necessity as preclinical models for testing of cell-based therapies in autoimmune diseases such as RA.

Dexamethasone and Monophosphoryl Lipid A Induce a Distinctive Profile on Monocyte-Derived Dendritic Cells through Transcriptional Modulation of Genes Associated With Essential Processes of the Immune Response.

There is growing interest in the use of tolerogenic dendritic cells (tolDCs) as a potential target for immunotherapy. However, the molecular bases that drive the differentiation of monocyte-derived DCs (moDCs) toward a tolerogenic state are still poorly understood. Here, we studied the transcriptional profile of moDCs from healthy subjects, modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), referred to as Dex-modulated and MPLA-activated DCs (DM-DCs), as an approach to identify molecular regulators and pathways associated with the induction of tolerogenic properties in tolDCs. We found that DM-DCs exhibit a distinctive transcriptional profile compared to untreated (DCs) and MPLA-matured DCs. Differentially expressed genes downregulated by DM included MMP12, CD1c, IL-1B, and FCER1A involved in DC maturation/inflammation and genes upregulated by DM included JAG1, MERTK, IL-10, and IDO1 involved in tolerance. Genes related to chemotactic responses, cell-to-cell signaling and interaction, fatty acid oxidation, metal homeostasis, and free radical scavenging were strongly enriched, predicting the activation of alternative metabolic processes than those driven by counterpart DCs. Furthermore, we identified a set of genes that were regulated exclusively by the combined action of Dex and MPLA, which are mainly involved in the control of zinc homeostasis and reactive oxygen species production. These data further support the important role of metabolic processes on the control of the DC-driven regulatory immune response. Thus, Dex and MPLA treatments modify gene expression in moDCs by inducing a particular transcriptional profile characterized by the activation of tolerance-associated genes and suppression of the expression of inflammatory genes, conferring the potential to exert regulatory functions and immune response modulation.

Peripheral T-Cell Reactivity to Heat Shock Protein 70 and Its Cofactor GrpE from Tropheryma whipplei Is Reduced in Patients with Classical Whipple's Disease.

Classical Whipple's disease (CWD) is characterized by the lack of specific Th1 response toward Tropheryma whipplei in genetically predisposed individuals. The cofactor GrpE of heat shock protein 70 (Hsp70) from T. whipplei was previously identified as a B-cell antigen. We tested the capacity of Hsp70 and GrpE to elicit specific proinflammatory T-cell responses. Peripheral mononuclear cells from CWD patients and healthy donors were stimulated with T. whipplei lysate or recombinant GrpE or Hsp70 before levels of CD40L, CD69, perforin, granzyme B, CD107a, and gamma interferon (IFN-γ) were determined in T cells by flow cytometry. Upon stimulation with total bacterial lysate or recombinant GrpE or Hsp70 of T. whipplei, the proportions of activated effector CD4+ T cells, determined as CD40L+ IFN-γ+, were significantly lower in patients with CWD than in healthy controls; CD8+ T cells of untreated CWD patients revealed an enhanced activation toward unspecific stimulation and T. whipplei-specific degranulation, although CD69+ IFN-γ+ CD8+ T cells were reduced upon stimulation with T. whipplei lysate and recombinant T. whipplei-derived proteins. Hsp70 and its cofactor GrpE are immunogenic in healthy individuals, eliciting effective responses against T. whipplei to control bacterial spreading. The lack of specific T-cell responses against these T. whipplei-derived proteins may contribute to the pathogenesis of CWD.

Treatment with Dexamethasone and Monophosphoryl Lipid A Removes Disease-Associated Transcriptional Signatures in Monocyte-Derived Dendritic Cells from Rheumatoid Arthritis Patients and Confers Tolerogenic Features.

Tolerogenic dendritic cells (TolDCs) are promising tools for therapy of autoimmune diseases, such as rheumatoid arthritis (RA). Here, we characterize monocyte-derived TolDCs from RA patients modulated with dexamethasone and activated with monophosphoryl lipid A (MPLA), referred to as MPLA-tDCs, in terms of gene expression, phenotype, cytokine profile, migratory properties, and T cell-stimulatory capacity in order to explore their suitability for cellular therapy. MPLA-tDCs derived from RA patients displayed an anti-inflammatory profile with reduced expression of co-stimulatory molecules and high IL-10/IL-12 ratio, but were capable of migrating toward the lymphoid chemokines CXCL12 and CCL19. These MPLA-tDCs induced hyporesponsiveness of autologous CD4+ T cells specific for synovial antigens in vitro. Global transcriptome analysis confirmed a unique transcriptional profile of MPLA-tDCs and revealed that RA-associated genes, which were upregulated in untreated DCs from RA patients, returned to expression levels of healthy donor-derived DCs after treatment with dexamethasone and MPLA. Thus, monocyte-derived DCs from RA patients have the capacity to develop tolerogenic features at transcriptional as well as at translational level, when modulated with dexamethasone and MPLA, overcoming disease-related effects. Furthermore, the ability of MPLA-tDCs to impair T cell responses to synovial antigens validates their potential as cellular treatment for RA.

Dexamethasone and Monophosphoryl Lipid A-Modulated Dendritic Cells Promote Antigen-Specific Tolerogenic Properties on Naive and Memory CD4+ T Cells.

Tolerogenic dendritic cells (DCs) are a promising tool to control T cell-mediated autoimmunity. Here, we evaluate the ability of dexamethasone-modulated and monophosphoryl lipid A (MPLA)-activated DCs [MPLA-tolerogenic DCs (tDCs)] to exert immunomodulatory effects on naive and memory CD4+ T cells in an antigen-specific manner. For this purpose, MPLA-tDCs were loaded with purified protein derivative (PPD) as antigen and co-cultured with autologous naive or memory CD4+ T cells. Lymphocytes were re-challenged with autologous PPD-pulsed mature DCs (mDCs), evaluating proliferation and cytokine production by flow cytometry. On primed-naive CD4+ T cells, the expression of regulatory T cell markers was evaluated and their suppressive ability was assessed in autologous co-cultures with CD4+ effector T cells and PPD-pulsed mDCs. We detected that memory CD4+ T cells primed by MPLA-tDCs presented reduced proliferation and proinflammatory cytokine expression in response to PPD and were refractory to subsequent stimulation. Naive CD4+ T cells were instructed by MPLA-tDCs to be hyporesponsive to antigen-specific restimulation and to suppress the induction of T helper cell type 1 and 17 responses. In conclusion, MPLA-tDCs are able to modulate antigen-specific responses of both naive and memory CD4+ T cells and might be a promising strategy to "turn off" self-reactive CD4+ effector T cells in autoimmunity.

Tolerogenic dendritic cells for reprogramming of lymphocyte responses in autoimmune diseases.

Dendritic cells (DCs) control immune responses by driving potent inflammatory actions against external and internal threats while generating tolerance to self and harmless components. This duality and their potential to reprogram immune responses in an antigen-specific fashion have made them an interesting target for immunotherapeutic strategies to control autoimmune diseases. Several protocols have been described for in vitro generation of tolerogenic DCs (tolDCs) capable of modulating adaptive immune responses and restoring tolerance through different mechanisms that involve anergy, generation of regulatory lymphocyte populations, or deletion of potentially harmful inflammatory T cell subsets. Recently, the capacity of tolDCs to induce interleukin (IL-10)-secreting regulatory B cells has been demonstrated. In vitro assays and rodent models of autoimmune diseases provide insights to the molecular regulators and pathways enabling tolDCs to control immune responses. Here we review mechanisms through which tolDCs modulate adaptive immune responses, particularly focusing on their suitability for reprogramming autoreactive CD4+ effector T cells. Furthermore, we discuss recent findings establishing that tolDCs also modulate B cell populations and discuss clinical trials applying tolDCs to patients with autoimmune diseases.

Gene Expression Profiling of Human Monocyte-derived Dendritic Cells - Searching for Molecular Regulators of Tolerogenicity.

The ability of dendritic cells (DCs) to initiate and modulate antigen-specific immune responses has made them attractive targets for immunotherapy. Since DC research in humans is limited by the scarcity of DC populations in the blood circulation, most of our knowledge about DC biology and function has been obtained in vitro from monocyte-derived DCs (moDCs), which can be readily generated in sufficient numbers and are able to differentiate into distinct functional subsets depending on the nature of stimulus. In particular, moDCs with tolerogenic properties (tolDCs) possess great therapeutic potential for the treatment of autoimmune diseases. Several protocols have been developed to generate tolDCs in vitro, able to reinstruct auto-reactive T cells and to promote regulatory cells. While ligands and soluble mediators, by which DCs shape immune responses, have been vastly studied, the intracellular pathways and transcriptional regulators that govern tolDC differentiation and function are poorly understood. Whole-genome microarrays and proteomics provide useful strategies to dissect the complex molecular processes that promote tolerogenicity. Only few attempts have been made to understand tolDC biology through a global view on "omics" profiles. So far, the identification of a common regulator of tolerogenicity has been hampered by the fact that each protocol, used for tolDC generation, targets distinct signaling pathways. Here, we review the progress in understanding the transcriptional regulation of moDC differentiation, with a special focus on tolDCs, and highlight candidate molecules that might be associated with DC tolerogenicity.

Therapeutic Potential of Hyporesponsive CD4(+) T Cells in Autoimmunity.

The interaction between dendritic cells (DCs) and T cells is crucial on immunity or tolerance induction. In an immature or semi-mature state, DCs induce tolerance through T-cell deletion, generation of regulatory T cells, and/or induction of T-cell anergy. Anergy is defined as an unresponsive state that retains T cells in an "off" mode under conditions in which immune activation is undesirable. This mechanism is crucial for the control of T-cell responses against self-antigens, thereby preventing autoimmunity. Tolerogenic DCs (tDCs), generated in vitro from peripheral blood monocytes of healthy donors or patients with autoimmune pathologies, were shown to modulate immune responses by inducing T-cell hyporesponsiveness. Animal models of autoimmune diseases confirmed the impact of T-cell anergy on disease development and progression in vivo. Thus, the induction of T-cell hyporesponsiveness by tDCs has become a promising immunotherapeutic strategy for the treatment of T-cell-mediated autoimmune disorders. Here, we review recent findings in the area and discuss the potential of anergy induction for clinical purposes.

Skewing dendritic cell differentiation towards a tolerogenic state for recovery of tolerance in rheumatoid arthritis.

To date, the available options to treat autoimmune diseases such as rheumatoid arthritis (RA) include traditional corticoids and biological drugs, which are not exempt of adverse effects. The development of cellular therapies based on dendritic cells with tolerogenic functions (TolDCs) has opened a new possibility to efficiently eradicate symptoms and control the immune response in the field of autoimmunity. TolDCs are an attractive tool for antigen-specific immunotherapy to restore self-tolerance in RA and other autoimmune disorders. A promising strategy is to inject autologous self-antigen-loaded TolDCs, which are able to delete or reprogram autoreactive T cells. Different protocols for the generation of stable human TolDCs have been established and the therapeutic effect of TolDCs has been investigated in multiple rodent models of arthritis. Pilot studies in humans confirmed that TolDC application is safe, encouraging clinical trials using self-antigen-loaded TolDCs in RA patients. Although an abundance of molecular regulators of DC functions has been discovered in the last decade, no master regulator of tolerogenicity has been identified yet. Further research is required to define biomarkers or key regulators of tolerogenicity that might facilitate the induction and monitoring of TolDCs.

Role of dendritic cells in the pathogenesis of Whipple's disease.

Accumulation of Tropheryma whipplei-stuffed macrophages in the duodenum, impaired T. whipplei-specific Th1 responses, and weak secretion of interleukin-12 (IL-12) are hallmarks of classical Whipple's disease (CWD). This study addresses dendritic cell (DC) functionality during CWD. We documented composition, distribution, and functionality of DC ex vivo or after in vitro maturation by fluorescence-activated cell sorting (FACS) and by immunohistochemistry in situ. A decrease in peripheral DC of untreated CWD patients compared to healthy donors was due to reduced CD11c(high) myeloid DC (M-DC). Decreased maturation markers CD83, CD86, and CCR7, as well as low IL-12 production in response to stimulation, disclosed an immature M-DC phenotype. In vitro-generated monocyte-derived DC from CWD patients showed normal maturation and T cell-stimulatory capacity under proinflammatory conditions but produced less IL-12 and failed to activate T. whipplei-specific Th1 cells. In duodenal and lymphoid tissues, T. whipplei was found within immature DC-SIGN(+) DC. DC and proliferating lymphocytes were reduced in lymph nodes of CWD patients compared to levels in controls. Our results indicate that dysfunctional IL-12 production by DC provides suboptimal conditions for priming of T. whipplei-specific T cells during CWD and that immature DC carrying T. whipplei contribute to the dissemination of the bacterium.

Effect of age on the CD4⁺ T-cell impairment in HIV-infected persons without and with cART.

BACKGROUND: Knowledge about HIV infection in older persons is becoming increasingly important. CD4⁺ T cells are essential for protective immunity, but little is known about the effect of age on the CD4⁺ T-cell impairment in HIV infection. METHODS: Treatment-naive patients aged older than 50 or younger than 40 years were studied for absolute and relative frequencies of CD31⁺ naive and CD31⁻ naive CD4⁺ T cells, central memory, effector memory, and terminally differentiated CD4⁺ T cells, and compared with age-matched controls. In addition, cellular proliferation and cytokine secretion properties were determined. CD4⁺ T-cell reconstitution was analyzed in older and younger patients with <350 or ≥ 350 CD4⁺ T cells per microliter at initiation of combination antiretroviral therapy (cART). RESULTS: CD4⁺ T cells of older but not younger HIV-infected patients showed age-inappropriate low levels of CD31⁻ naive cells, increased levels of effector memory cells, and enhanced interferonγ and interleukin-17 secretion. Impaired CD4⁺ T-cell composition persisted in patients who initiated cART at <350 CD4⁺ T cells per microliter. In patients with CD4⁺ T cells ≥ 350 per microliter, alterations were less pronounced and were reversible with cART. Compared with age-matched controls, total CD4⁺ T-cell counts did not differ between treated younger and older HIV-infected patients. CONCLUSIONS: These data demonstrate that aging enhances the CD4⁺ T-cell impairment in HIV-infected persons mainly by a loss of CD31⁻ naive cells, accumulation of effector memory cells, and increased pro-inflammatory effector functions. Age-related changes in CD4⁺ T-cell composition can be prevented by an early initiation of cART.

Macrophages accumulate in the gut mucosa of untreated HIV-infected patients.

BACKGROUND: Mucosal macrophages are involved in the maintenance of epithelial barrier integrity and the elimination of invading pathogens. Although an intestinal barrier defect and microbial translocation are hallmarks of human immunodeficiency virus (HIV) infection, recent data on gut mucosal macrophages in HIV infection are sparse. METHODS: Treatment-naive and treated HIV-infected patients and healthy controls were studied for frequencies and functional parameters of blood monocytes and macrophages in duodenal mucosa. RESULTS: We found mucosal enrichment of macrophages in untreated HIV infection associated with reduced monocyte counts in blood and increased monocyte expression of the gut-homing molecule integrin ß7. Increased CCR2 density on integrin ß7-expressing monocytes and mucosal secretion of CCL2 suggest that CCR2/CCL2-chemotaxis is involved in enhanced trafficking of blood monocytes to the gut. Secretion of macrophage-related proinflammatory molecules interleukin 1ß, CCL5, CXCL9, and CXCL10 was increased in the gut mucosa of untreated patients. Moreover, mucosal macrophages of untreated patients showed reduced phagocytic activity. CONCLUSIONS: These data suggest a role for gut mucosal macrophages in HIV immune pathogenesis: infiltrated macrophages in the intestinal mucosa may promote local inflammation and tissue injury, whereas their low phagocytic activity prevents the efficient elimination of luminal antigens that cross the damaged intestinal barrier.

Evaluation of arginine metabolism for the analysis of M1/M2 macrophage activation in human clinical specimens.

BACKGROUND: Macrophage heterogeneity reflects their plasticity in response to environmental stimuli. Usually human macrophages are characterized by analysis of surface molecules or cytokine expression while functional assays are established in the mouse system but lacking for various human specimens. METHODS: To evaluate the value of analysis of arginine metabolism for characterization of human macrophage differentiation, we analyzed nitrite production and arginase activity in plasma, duodenal biopsies, and in vitro differentiated macrophages of patients with classical Whipple's disease. RESULTS: We demonstrate that it is feasible to determine the content of urea in supernatants of stimulated duodenal biopsies, arginase activity in fresh duodenal biopsies and plasma samples, and arginase activity and nitrite production in lysates and supernatants of in vitro differentiated macrophages. However, only selected tests are appropriate to define macrophage polarization in human specimens. CONCLUSION: Analysis of arginine metabolism is not suitable for the characterization of in vitro differentiated human macrophages. Besides the measurement of nitrite in duodenal biopsy supernatants, the determination of arginase activity in human plasma seems to be a reasonable functional test to detect enhanced M2 macrophage activation and, thus, is of great value for the analysis of macrophage activity with a minimum of material and costs.

Immunopathology of immune reconstitution inflammatory syndrome in Whipple's disease.

During antimicrobial treatment of classic Whipple's disease (CWD), the chronic systemic infection with Tropheryma whipplei, immune reconstitution inflammatory syndrome (IRIS), is a serious complication. The aim of our study was to characterize the immunological processes underlying IRIS in CWD. Following the definition of IRIS, we describe histological features of IRIS and immunological parameters of 24 CWD IRIS patients, 189 CWD patients without IRIS, and 89 healthy individuals. T cell reconstitution, Th1 reactivity, and the phenotype of T cells were described in the peripheral blood, and infiltration of CD4(+) T cells and regulatory T cells in the duodenal mucosa was determined. During IRIS, tissues were heavily infiltrated by CD3(+), predominantly CD45RO(+)CD4(+) T cells. In the periphery, initial reduction of CD4(+) cell counts and their reconstitution on treatment was more pronounced in CWD patients with IRIS than in those without IRIS. The ratio of activated and regulatory CD4(+) T cells, nonspecific Th1 reactivity, and the proportion of naive among CD4(+) T cells was high, whereas serum IL-10 was low during IRIS. T. whipplei-specific Th1 reactivity remained suppressed before and after emergence of IRIS. The findings that IRIS in CWD mainly are mediated by nonspecific activation of CD4(+) T cells and that it is not sufficiently counterbalanced by regulatory T cells indicate that flare-up of pathogen-specific immunoreactivity is not instrumental in the pathogenesis of IRIS in CWD.

Regulatory T cells in patients with Whipple's disease.

Classical Whipple's disease (CWD) is caused by chronic infection with Tropheryma whipplei that seems to be associated with an underlying immune defect. The pathognomonic hallmark of CWD is a massive infiltration of the duodenal mucosa with T. whipplei-infected macrophages that disperse systemically to many other organ systems. An alleviated inflammatory reaction and the absence of T. whipplei-specific Th1 reactivity support persistence and systemic spread of the pathogen. In this article, we hypothesized that regulatory T cells (T(reg)) are involved in immunomodulation in CWD, and we asked for the distribution, activation, and regulatory capacity of T(reg) in CWD patients. Whereas in the lamina propria of CWD patients before treatment numbers of T(reg) were increased, percentages in the peripheral blood were similar in CWD patients and healthy controls. However, peripheral T(reg) of CWD patients were more activated than those of controls. Elevated secretion of IL-10 and TGF-ß in the duodenal mucosa of CWD patients indicated locally enhanced T(reg) activity. Enhanced CD95 expression on peripheral memory CD4(+) T cells combined with reduced expression of IFN-γ and IL-17A upon polyclonal stimulation by CD4(+) cells from untreated CWD patients further hinted to T(reg) activity-related exhaustion of effector CD4(+) T cells. In conclusion, increased numbers of T(reg) can be detected within the duodenal mucosa in untreated CWD, where huge numbers of T. whipplei-infected macrophages are present. Thus, T(reg) might contribute to the chronic infection and systemic spread of T. whipplei in CWD but in contrast prevent mucosal barrier defect by reducing local inflammation.