Humoral immunity in tuberculin skin test anergy and its role in high-risk persons exposed to active tuberculosis.

The most common test to identify latent tuberculosis is the tuberculin skin test that detects T cell responses of delayed type hypersensitivity type IV. Since it produces false negative reactions in active tuberculosis or in high-risk persons exposed to tuberculosis patients as shown in this report, we studied antibody profiles to explain the anergy of such responses in high-risk individuals without active infection. Our results showed that humoral immunity against tuberculin, regardless of the result of the tuberculin skin test is important for protection from active tuberculosis and that the presence of high antibody titers is a more reliable indicator of infection latency suggesting that latency can be based on the levels of antibodies together with in vitro proliferation of peripheral blood mononuclear cells in the presence of the purified protein derivative. Importantly, anti-tuberculin IgG antibody levels mediate the anergy described herein, which could also prevent reactivation of disease in high-risk individuals with high antibody titers. Such anti-tuberculin IgG antibodies were also found associated with blocking and/or stimulation of in vitro cultures of PBMC with tuberculin. In this regard, future studies need to establish if immune responses to Mycobacterium tuberculosis can generate a broad spectrum of reactions either toward Th1 responses favoring stimulation by cytokines or by antibodies and those toward diminished responses by Th2 cytokines or blocking by antibodies; possibly involving mechanisms of antibody dependent protection from Mtb by different subclasses of IgG.

Localized diacylglycerol-dependent stimulation of Ras and Rap1 during phagocytosis.

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcgamma receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcgamma receptor-mediated phagocytosis to enhance the innate immune response.

Maturation of lymph node fibroblastic reticular cells from myofibroblastic precursors is critical for antiviral immunity.

The stromal scaffold of the lymph node (LN) paracortex is built by fibroblastic reticular cells (FRCs). Conditional ablation of lymphotoxin-β receptor (LTβR) expression in LN FRCs and their mesenchymal progenitors in developing LNs revealed that LTβR-signaling in these cells was not essential for the formation of LNs. Although T cell zone reticular cells had lost podoplanin expression, they still formed a functional conduit system and showed enhanced expression of myofibroblastic markers. However, essential immune functions of FRCs, including homeostatic chemokine and interleukin-7 expression, were impaired. These changes in T cell zone reticular cell function were associated with increased susceptibility to viral infection. Thus, myofibroblasic FRC precursors are able to generate the basic T cell zone infrastructure, whereas LTβR-dependent maturation of FRCs guarantees full immunocompetence and hence optimal LN function during infection.

Nuclear localization of HBD-1 in human keratinocytes.

OBJECTIVE: Human defensins and cathelicidins are a family of cationic antimicrobial peptides (AMPs), which play multiple roles in both innate and adaptive immune systems. They have direct antimicrobial activity against several microorganisms including burn pathogens. The majority of components of innate and adaptive immunity either express naturally occurring defensins or are otherwise chemoattracted or functionally affected by them. They also enhance adaptive immunity and wound healing and alter antibody production. All mechanisms to explain multiple functions of AMPs are not clearly understood. Prior studies to localize defensins in normal and burned skin using deconvolution fluorescence scanning microscopy indicate localization of defensins in the nucleus, perinuclear regions, and cytoplasm. The objective of this study is to further confirm the identification of HBD-1 in the nucleus by deconvolution microscopic studies involving image reconstruction and wire frame modeling. RESULTS: Our study demonstrated the presence of intranuclear HBD-1 in keratinocytes throughout the stratum spinosum by costaining with the nuclear probe DAPI. In addition, HBD-1 sequence does show some homology with known cationic nuclear localization signal sequences. CONCLUSION: To our knowledge, this is the first report to localize HBD-1 in the nuclear region, suggesting a role for this peptide in gene expression and providing new data that may help determine mechanisms of defensin functions.

Highly pathogenic adapted HIV-1 strains limit host immunity and dictate rapid disease progression.

OBJECTIVE: The study of HIV-1 rapid progressors has been limited to specific case reports. Nevertheless, identification and characterization of the viral and host factors involved in rapid progression are crucial when attempting to uncover the correlates of rapid disease outcome. DESIGN: We carried out comparative functional analyses in rapid progressors (n = 46) and standard progressors (n = 46) early after HIV-1 seroconversion (≤1 year). The viral traits tested were viral replicative capacity, co-receptor usage, and genomic variation. Host CD8 T-cell responses, humoral activity, and HLA immunogenetic markers were also determined. RESULTS: Our data demonstrate an unusual convergence of highly pathogenic HIV-1 strains in rapid progressors. Compared with standard progressors, rapid progressor viral strains show higher in-vitro replicative capacity (81.5 vs. 67.9%; P = 0.025) and greater X4/DM co-receptor usage (26.3 vs. 2.8%; P = 0.006) in early infection. Limited or absent functional HIV-1 CD8 T-cell responses and neutralizing activity were measured in rapid progressors. Moreover, the increase in common HLA allele-restricted CD8 T-cell escape mutations in rapid progressors acts as a signature of uncontrolled HIV-1 replication and early impairment of adaptive cellular responses. CONCLUSION: Our data support a dominant role for viral factors in rapid progressors. Robust HIV-1 replication and intrinsic viral properties limit host adaptive immune responses, thus driving rapid disease progression.

Expression and function of psoriasin (S100A7) and koebnerisin (S100A15) in the brain

The expression and function of psoriasin in the brain have been insufficiently characterized. Here, we show the induction of psoriasin expression in the central nervous system (CNS) after bacterial and viral stimulation. We used a pneumococcal meningitis in vivo model that revealed S100A15 expression in astrocytes and meningeal cells. These results were confirmed by a cell-based in vivo assay using primary rat glial and meningeal cell cultures. We investigated psoriasin expression in glial and meningeal cells using polyinosinic-polycytidylic acid, a synthetic analog of double-stranded RNA that mimics viral infection. Furthermore, previous results showed that antimicrobial peptides have not only bactericidal but also immunomodulatory functions. To test this statement, we used recombinant psoriasin as a stimulus. Glial and meningeal cells were treated with recombinant psoriasin at concentrations from 25 to 500 ng/ml. Treated microglia and meningeal cells showed phosphorylation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2 (ERK1/2) signal transduction pathway. We demonstrated that this activation of ERK depends on RAGE, the receptor for advanced glycation end products. Furthermore, microglia cells treated with recombinant psoriasin change their phenotype to an enlarged shape. In conclusion, our results indicate an occurrence of psoriasin in the brain. An involvement of psoriasin as an antimicrobial protein that modulates the innate immune system after bacterial or viral stimulation is possible.

Additive effects of HLA alleles and innate immune genes determine viral outcome in HCV infection

BACKGROUND Chronic HCV infection is a leading cause of liver-related morbidity globally. The innate and adaptive immune responses are thought to be important in determining viral outcomes. Polymorphisms associated with the IFNL3 (IL28B) gene are strongly associated with spontaneous clearance and treatment outcomes. OBJECTIVE This study investigates the importance of HLA genes in the context of genetic variation associated with the innate immune genes IFNL3 and KIR2DS3. DESIGN We assess the collective influence of HLA and innate immune genes on viral outcomes in an Irish cohort of women (n=319) who had been infected from a single source as well as a more heterogeneous cohort (Swiss Cohort, n=461). In the Irish cohort, a number of HLA alleles are associated with different outcomes, and the impact of IFNL3-linked polymorphisms is profound. RESULTS Logistic regression was performed on data from the Irish cohort, and indicates that the HLA-A*03 (OR 0.36 (0.15 to 0.89), p=0.027) -B*27 (OR 0.12 (0.03 to 0.45), p=<0.001), -DRB1*01:01 (OR 0.2 (0.07 to 0.61), p=0.005), -DRB1*04:01 (OR 0.31 (0.12 to 0.85, p=0.02) and the CC IFNL3 rs12979860 genotypes (OR 0.1 (0.04 to 0.23), p<0.001) are significantly associated with viral clearance. Furthermore, DQB1*02:01 (OR 4.2 (2.04 to 8.66), p=0.008), KIR2DS3 (OR 4.36 (1.62 to 11.74), p=0.004) and the rs12979860 IFNL3 'T' allele are associated with chronic infection. This study finds no interactive effect between IFNL3 and these Class I and II alleles in relation to viral clearance. There is a clear additive effect, however. Data from the Swiss cohort also confirms independent and additive effects of HLA Class I, II and IFNL3 genes in their prediction of viral outcome. CONCLUSIONS This data supports a critical role for the adaptive immune response in the control of HCV in concert with the innate immune response.

Potent innate immune response to pathogenic leptospira in human whole blood.

BACKGROUND Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. The bacteria enter the human body via abraded skin or mucous membranes and may disseminate throughout. In general the clinical picture is mild but some patients develop rapidly progressive, severe disease with a high case fatality rate. Not much is known about the innate immune response to leptospires during haematogenous dissemination. Previous work showed that a human THP-1 cell line recognized heat-killed leptospires and leptospiral LPS through TLR2 instead of TLR4. The LPS of virulent leptospires displayed a lower potency to trigger TNF production by THP-1 cells compared to LPS of non-virulent leptospires. METHODOLOGY/PRINCIPAL FINDINGS We investigated the host response and killing of virulent and non-virulent Leptospira of different serovars by human THP-1 cells, human PBMC's and human whole blood. Virulence of each leptospiral strain was tested in a well accepted standard guinea pig model. Virulent leptospires displayed complement resistance in human serum and whole blood while in-vitro attenuated non-virulent leptospires were rapidly killed in a complement dependent manner. In vitro stimulation of THP-1 and PBMC's with heat-killed and living leptospires showed differential serovar and cell type dependence of cytokine induction. However, at low, physiological, leptospiral dose, living virulent complement resistant strains were consistently more potent in whole blood stimulations than the corresponding non-virulent complement sensitive strains. At higher dose living virulent and non-virulent leptospires were equipotent in whole blood. Inhibition of different TLRs indicated that both TLR2 and TLR4 as well as TLR5 play a role in the whole blood cytokine response to living leptospires. CONCLUSIONS/SIGNIFICANCE Thus, in a minimally altered system as human whole blood, highly virulent Leptospira are potent inducers of the cytokine response.

Innate immune cells express IL-17A/F in acute generalized exanthematous pustulosis and generalized pustular psoriasis.

Acute generalized exanthematous pustulosis (AGEP) and generalized pustular psoriasis (GPP) are rare pustular skin disorders with systemic involvement. IL-17A/F is a proinflammatory cytokine involved in various neutrophilic inflammatory disorders. Here we show that IL-17A/F is highly expressed by innate immune cells such as neutrophils and mast cells in both AGEP and GPP.

BID-dependent release of mitochondrial SMAC dampens XIAP-mediated immunity against Shigella

The X‐linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti‐apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase‐mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP‐mediated immune response by inducing the BID‐dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain‐dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.

Microbiota-derived compounds drive steady-state granulopoiesis via MyD88/TICAM signaling.

Neutropenia is probably the strongest known predisposition to infection with otherwise harmless environmental or microbiota-derived species. Because initial swarming of neutrophils at the site of infection occurs within minutes, rather than the hours required to induce "emergency granulopoiesis," the relevance of having high numbers of these cells available at any one time is obvious. We observed that germ-free (GF) animals show delayed clearance of an apathogenic bacterium after systemic challenge. In this article, we show that the size of the bone marrow myeloid cell pool correlates strongly with the complexity of the intestinal microbiota. The effect of colonization can be recapitulated by transferring sterile heat-treated serum from colonized mice into GF wild-type mice. TLR signaling was essential for microbiota-driven myelopoiesis, as microbiota colonization or transferring serum from colonized animals had no effect in GF MyD88(-/-)TICAM1(-/-) mice. Amplification of myelopoiesis occurred in the absence of microbiota-specific IgG production. Thus, very low concentrations of microbial Ags and TLR ligands, well below the threshold required for induction of adaptive immunity, sets the bone marrow myeloid cell pool size. Coevolution of mammals with their microbiota has probably led to a reliance on microbiota-derived signals to provide tonic stimulation to the systemic innate immune system and to maintain vigilance to infection. This suggests that microbiota changes observed in dysbiosis, obesity, or antibiotic therapy may affect the cross talk between hematopoiesis and the microbiota, potentially exacerbating inflammatory or infectious states in the host.

Basophils promote innate lymphoid cell responses in inflamed skin.

Type 2 inflammation underlies allergic diseases such as atopic dermatitis, which is characterized by the accumulation of basophils and group 2 innate lymphoid cells (ILC2s) in inflamed skin lesions. Although murine studies have demonstrated that cutaneous basophil and ILC2 responses are dependent on thymic stromal lymphopoietin, whether these cell populations interact to regulate the development of cutaneous type 2 inflammation is poorly defined. In this study, we identify that basophils and ILC2s significantly accumulate in inflamed human and murine skin and form clusters not observed in control skin. We demonstrate that murine basophil responses precede ILC2 responses and that basophils are the dominant IL-4-enhanced GFP-expressing cell type in inflamed skin. Furthermore, basophils and IL-4 were necessary for the optimal accumulation of ILC2s and induction of atopic dermatitis-like disease. We show that ILC2s express IL-4Rα and proliferate in an IL-4-dependent manner. Additionally, basophil-derived IL-4 was required for cutaneous ILC2 responses in vivo and directly regulated ILC2 proliferation ex vivo. Collectively, these data reveal a previously unrecognized role for basophil-derived IL-4 in promoting ILC2 responses during cutaneous inflammation.

Multiple roles of complement MASP-1 at the interface of innate immune response and coagulation

MASP-1 is a versatile serine protease that cleaves a number of substrates in human blood. In recent years it became evident that besides playing a crucial role in complement activation MASP-1 also triggers other cascade systems and even cells to mount a more powerful innate immune response. In this review we summarize the latest discoveries about the diverse functions of this multi-faceted protease. Recent studies revealed that among MBL-associated serine proteases, MASP-1 is the one responsible for triggering the lectin pathway via its ability to rapidly autoactivate then cleave MASP-2, and possibly MASP-3. The crystal structure of MASP-1 explains its more relaxed substrate specificity compared to the related complement enzymes. Due to the relaxed specificity, MASP-1 interacts with the coagulation cascade and the kinin generating system, and it can also activate endothelial cells eliciting pro-inflammatory signaling.