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.

Sequential treatment of progressive multifocal leukoencephalopathy with intravenous immunoglobulins and pembrolizumab.

Progressive multifocal leukoencephalopathy (PML) is a rare demyelinating disease of the CNS caused by the human polyomavirus 2 (JCV). PML predominantly occurs in immunocompromised patients. To date, no specific antiviral treatment exists, leaving only restoration of the immune system as possible treatment. In 2019, the monoclonal antibody pembrolizumab was reported as a potential treatment option in PML in a case series. Following case reports could not thoroughly confirm a positive outcome. Pembrolizumab targets the inhibitory programmed cell death protein 1 (PD-1) receptor on lymphocytes and is associated with beneficial expansion of pre-existing virus-specific T cells. Here we describe a patient with PML who benefited from combined treatment with intravenous immunoglobulins, maraviroc, and pembrolizumab.

T Cell-Dependent Maturation of Pathogen-Specific Igs in the Antrum of Chronically Helicobacter pylori-Infected Patients.

Mucosal plasma cells (PC) and Ig production are essential to fend pathogens and to maintain mucosal homeostasis. In human Helicobacter pylori infection, mucosal PC express inducible NO synthase (iNOS), which positively correlates with clearance of experimental human infection. To characterize Ig genes and specificities of antral mucosal iNOS+ and iNOS- PC in H. pylori infection, we sequenced rearranged Ig genes from single cell-sorted PC from biopsy specimens of chronically infected patients and analyzed them with respect to their molecular features. The binding specificity of individual PC's Ig was determined following recombinant expression. We identified high rates of somatic hypermutations, especially targeting RGYW/WRCY hotspot motifs in the individual Ig genes, indicating T cell-dependent maturation. For seven of 14 recombinantly expressed Ig, Ag specificity could be determined. Two clones reacted to H. pylori proteins, and five were found to be polyreactive against LPSs, dsDNA, and ssDNA. All specific Ig originated from iNOS+ PC. H. pylori-specific Ig are encoded by V and J family genes previously shown to be also used in rearranged Ig loci of MALT B cell lymphomas. In summary, mucosal iNOS+ PC producing H. pylori-specific Ig accumulate in infection and appear to be a product of T cell-dependent B cell maturation. Moreover, the Ig's molecular features partly resembled that of MALT B cell lymphoma Ig genes, suggestive of a mechanism in which a progressive molecular evolution of pathogen-specific B cells to MALT B cell lymphoma occurs.

Mucosal Inducible NO Synthase-Producing IgA+ Plasma Cells in Helicobacter pylori-Infected Patients.

The mucosal immune system is relevant for homeostasis, immunity, and also pathological conditions in the gastrointestinal tract. Inducible NO synthase (iNOS)-dependent production of NO is one of the factors linked to both antimicrobial immunity and pathological conditions. Upregulation of iNOS has been observed in human Helicobacter pylori infection, but the cellular sources of iNOS are ill defined. Key differences in regulation of iNOS expression impair the translation from mouse models to human medicine. To characterize mucosal iNOS-producing leukocytes, biopsy specimens from H. pylori-infected patients, controls, and participants of a vaccination trial were analyzed by immunohistochemistry, along with flow cytometric analyses of lymphocytes for iNOS expression and activity. We newly identified mucosal IgA-producing plasma cells (PCs) as one major iNOS(+) cell population in H. pylori-infected patients and confirmed intracellular NO production. Because we did not detect iNOS(+) PCs in three distinct infectious diseases, this is not a general feature of mucosal PCs under conditions of infection. Furthermore, numbers of mucosal iNOS(+) PCs were elevated in individuals who had cleared experimental H. pylori infection compared with those who had not. Thus, IgA(+) PCs expressing iNOS are described for the first time, to our knowledge, in humans. iNOS(+) PCs are induced in the course of human H. pylori infection, and their abundance seems to correlate with the clinical course of the infection.

Specific CD4+ T-Cell Reactivity and Cytokine Release in Different Clinical Presentations of Leptospirosis.

Clinical manifestations of leptospirosis are highly variable: from asymptomatic to severe and potentially fatal. The outcome of the disease is usually determined in the immunological phase, beginning in the second week of symptoms. The underlying mechanisms, predictive factors, and individual immune responses that contribute to clinical variations are not well understood. The aim of this study was to determine the specifics of CD4(+) T-cell reactivity and cytokine release after stimulation with leptospiral antigens in patients with leptospirosis of different disease severities (patients with mild and severe symptoms) and in control subjects (with and without proven exposure to Leptospira). Whole-blood specimens were stimulated with Leptospira antigens in vitro. Subsequently, intracellular staining of cytokines was performed, and flow cytometry was used to assess the expression of CD40 ligand (CD40L) and the production of gamma interferon (IFN-γ), interleukin-10 (IL-10), IL-2, and tumor necrosis factor alpha (TNF-α) by CD4(+) T cells. The production of inflammatory cytokines such as TNF-α by CD4(+) T cells after stimulation with leptospiral antigens was highest in patients with severe disease. In contrast, the ratio of IL-10 production to TNF-α production was higher in exposed subjects than in patients with mild and severe disease. Levels of proinflammatory cytokines such as TNF-α may be useful markers of the severity of the immunological phase of leptospirosis. IL-10 production by T cells after antigen-specific stimulation may indicate a more successful downregulation of the inflammatory response and may contribute to an asymptomatic course of the disease.

CCR5Δ32 mutation and HIV infection: basis for curative HIV therapy.

The C-C chemokine receptor 5 (CCR5) is expressed on potential human immunodeficiency virus (HIV) target cells and serves as the predominant co-receptor for viral entry during initial transmission and through the early stages of infection. A homozygous Δ32 mutation in the CCR5 gene prevents CCR5 cell surface expression and thus confers resistance to infection with CCR5-tropic HIV strains. Transplantation of hematopoietic stem cells from a CCR5Δ32/Δ32 donor was previously successful in eliminating HIV from the recipient's immune system, suggesting that targeted CCR5 disruption can lead to an HIV cure. Therefore, intense work is currently being carried out on CCR5 gene-editing tools to develop curative HIV therapy. Here, we review the natural function of CCR5, the progress made on CCR5 gene editing to date and discuss the current limitations.

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.

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.

The Attenuated Brucella abortus Strain 19 Invades, Persists in, and Activates Human Dendritic Cells, and Induces the Secretion of IL-12p70 but Not IL-23.

BACKGROUND: Bacterial vectors have been proposed as novel vaccine strategies to induce strong cellular immunity. Attenuated strains of Brucella abortus comprise promising vector candidates since they have the potential to induce strong CD4(+) and CD8(+) T-cell mediated immune responses in the absence of excessive inflammation as observed with other Gram-negative bacteria. However, some Brucella strains interfere with the maturation of dendritic cells (DCs), which is essential for antigen-specific T-cell priming. In the present study, we investigated the interaction of human monocyte-derived DCs with the smooth attenuated B. abortus strain (S) 19, which has previously been employed successfully to vaccinate cattle. METHODOLOGY/PRINCIPAL FINDINGS: We first looked into the potential of S19 to hamper the cytokine-induced maturation of DCs; however, infected cells expressed CD25, CD40, CD80, and CD86 to a comparable extent as uninfected, cytokine-matured DCs. Furthermore, S19 activated DCs in the absence of exogeneous stimuli, enhanced the expression of HLA-ABC and HLA-DR, and was able to persist intracellularly without causing cytotoxicity. Thus, DCs provide a cellular niche for persisting brucellae in vivo as a permanent source of antigen. S19-infected DCs produced IL-12/23p40, IL-12p70, and IL-10, but not IL-23. While heat-killed bacteria also activated DCs, soluble mediators were not involved in S19-induced activation of human DCs. HEK 293 transfectants revealed cellular activation by S19 primarily through engagement of Toll-like receptor (TLR)2. CONCLUSIONS/SIGNIFICANCE: Thus, as an immunological prerequisite for vaccine efficacy, B. abortus S19 potently infects and potently activates (most likely via TLR2) human DCs to produce Th1-promoting cytokines.

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.

Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem cell transplantation.

HIV entry into CD4(+) cells requires interaction with a cellular receptor, generally either CCR5 or CXCR4. We have previously reported the case of an HIV-infected patient in whom viral replication remained absent despite discontinuation of antiretroviral therapy after transplantation with CCR5Δ32/Δ32 stem cells. However, it was expected that the long-lived viral reservoir would lead to HIV rebound and disease progression during the process of immune reconstitution. In the present study, we demonstrate successful reconstitution of CD4(+) T cells at the systemic level as well as in the gut mucosal immune system after CCR5Δ32/Δ32 stem cell transplantation, while the patient remains without any sign of HIV infection. This was observed although recovered CD4(+) T cells contain a high proportion of activated memory CD4(+) T cells, ie, the preferential targets of HIV, and are susceptible to productive infection with CXCR4-tropic HIV. Furthermore, during the process of immune reconstitution, we found evidence for the replacement of long-lived host tissue cells with donor-derived cells, indicating that the size of the viral reservoir has been reduced over time. In conclusion, our results strongly suggest that cure of HIV has been achieved in this patient.

Specific and nonspecific B-cell function in the small intestines of patients with Whipple's disease.

Whipple's disease is a chronic multisystemic infection caused by Tropheryma whipplei that is characterized by arthritis, weight loss, and diarrhea. The immunological defects in the duodenal mucosa, the site of major replication of the agent underlying the pathogenesis of Whipple's disease, are poorly understood. Mucosal immunoglobulins are essential for the defense against intestinal pathogens; therefore, we analyzed the B-cell response in duodenal specimens and sera of Whipple's disease patients. Whereas systemic immunoglobulin production was affected only marginally, duodenal biopsy specimens of Whipple's disease patients contained reduced numbers of immunoglobulin-positive plasma cells and secreted less immunoglobulin compared to healthy controls but showed a weak secretory IgA response toward T. whipplei. This T. whipplei-specific intestinal immune response was not observed in controls. Thus, we were able to demonstrate that general mucosal immunoglobulin production in Whipple's disease patients is impaired. However, this deficiency does not completely abolish T. whipplei-specific secretory IgA production that nonetheless does not protect from chronic infection.

Impaired immune functions of monocytes and macrophages in Whipple's disease.

BACKGROUND & AIMS: Whipple's disease is a chronic multisystemic infection caused by Tropheryma whipplei. Host factors likely predispose for the establishment of an infection, and macrophages seem to be involved in the pathogenesis of Whipple's disease. However, macrophage activation in Whipple's disease has not been studied systematically so far. METHODS: Samples from 145 Whipple's disease patients and 166 control subjects were investigated. We characterized duodenal macrophages and lymphocytes immunohistochemically and peripheral monocytes by flow cytometry and quantified mucosal and systemic cytokines and chemokines indicative for macrophage activation. In addition, we determined duodenal nitrite production and oxidative burst induced by T whipplei and by other bacteria. RESULTS: Reduced numbers of duodenal lymphocytes, increased numbers of CD163(+) and stabilin-1(+), reduced numbers of inducible nitric synthase+ duodenal macrophages, and increased percentages of CD163(+) peripheral monocytes indicated a lack of inflammation and a M2/alternatively activated macrophage phenotype in Whipple's disease. Incubation with T whipplei in vitro enhanced the expression of CD163 on monocytes from Whipple's disease patients but not from control subjects. Chemokines and cytokines associated with M2/alternative macrophage activation were elevated in the duodenum and the peripheral blood from Whipple's disease patients. Functionally, Whipple's disease patients showed a reduced duodenal nitrite production and reduced oxidative burst upon incubation with T whipplei compared with healthy subjects. CONCLUSIONS: The lack of excessive local inflammation and alternative activation of macrophages, triggered in part by the agent T whipplei itself, may explain the hallmark of Whipple's disease: invasion of the intestinal mucosa with macrophages incompetent to degrade T whipplei.

Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation.

Infection with the human immunodeficiency virus type 1 (HIV-1) requires the presence of a CD4 receptor and a chemokine receptor, principally chemokine receptor 5 (CCR5). Homozygosity for a 32-bp deletion in the CCR5 allele provides resistance against HIV-1 acquisition. We transplanted stem cells from a donor who was homozygous for CCR5 delta32 in a patient with acute myeloid leukemia and HIV-1 infection. The patient remained without viral rebound 20 months after transplantation and discontinuation of antiretroviral therapy. This outcome demonstrates the critical role CCR5 plays in maintaining HIV-1 infection.

Oral polio vaccination leads to oligoclonal expansion of TCRBV16+ and TCRBV13+ T cells in the colon of rhesus macaques.

In studying immune responses towards the poliovirus, data about T cell mediated immunity in the intestine as the main portal of viral entry in disease and vaccination is lacking. We treated two macaques with oral Polio vaccine and collected duodenal and colonic biopsy specimens. RNA isolation, reverse transcription, and polymerase chain reaction were performed for fragment analysis of the complementarity determining region 3 (CDR3) of the T cell receptor beta chain variable region (TCRBV), followed by subcloning and sequencing of expanded bands. In the colon, oligoclonal expansions of TCRBV16+ or TCRBV13+ intestinal T cells with conserved motifs of the hypervariable CDR3 were found. Flow cytometric analysis of mucosal T cells revealed that activated colonic T cells were mainly CD4(+). Our results indicate that there is a local activation of oligoclonal T cells in the colon after oral Polio vaccination (OPV) which involves selected TCRBV families and may occur within the CD4(+) T cell subset.

Migration patterns of nonspecifically activated versus nonactivated nonhuman primate T lymphocytes: preferential homing of activated autologous CD8+ T cells in the rectal mucosa.

Adoptive cell transfer may be a successful strategy in anticancer therapy and its therapeutic efficiency depends on the access of transferred cells to the tumor site and their persistence in vivo. Nevertheless, the migration properties of autologous in vitro-activated T cells in primates are largely unknown. Here, we established the long-term tracking of T-cell migration into various compartments of rhesus macaques as a preclinical model for the evaluation of T-cell-based immunotherapy. Peripheral blood mononuclear cells from 3 to 4 rhesus macaques were activated with anti-CD3/anti-CD28 or not, labeled with carboxyfluorescein diacetat succinimidyl ester, and reinjected intravenously into the donor animals. Blood samples, lymph node biopsies, and mucosal biopsies (duodenum, rectum) were collected at various time points and analyzed by flow cytometry for the presence of the reinjected T cells. We demonstrate that nonspecific in vitro activation changes the in vivo migratory behavior of T cells and provokes a preferential migration of CD8 T cells to the rectum. Nonspecifically activated transferred CD4 T cells were found in much lower frequencies at this site and also in other compartments. Thus, our results indicate an imbalanced distribution of autologous CD8 and CD4 T cells in various compartments that is more apparent when T cells are activated before the transfer. The migratory behavior of in vitro-expanded, autologously transferred T cells can, therefore, influence the clinical outcome of adoptive cell transfer.