Toll-like receptor 10 is involved in induction of innate immune responses to influenza virus infection.

Toll-like receptors (TLRs) play key roles in innate immune recognition of pathogen-associated molecular patterns of invading microbes. Among the 10 TLR family members identified in humans, TLR10 remains an orphan receptor without known agonist or function. TLR10 is a pseudogene in mice and mouse models are noninformative in this regard. Using influenza virus infection in primary human peripheral blood monocyte-derived macrophages and a human monocytic cell line, we now provide previously unidentified evidence that TLR10 plays a role in innate immune responses following viral infection. Influenza virus infection increased TLR10 expression and TLR10 contributed to innate immune sensing of viral infection leading to cytokine induction, including proinflammatory cytokines and interferons. TLR10 induction is more pronounced following infection with highly pathogenic avian influenza H5N1 virus compared with a low pathogenic H1N1 virus. Induction of TLR10 by virus infection requires active virus replication and de novo protein synthesis. Culture supernatants of virus-infected cells modestly up-regulate TLR10 expression in nonvirus-infected cells. Signaling via TLR10 was activated by the functional RNA-protein complex of influenza virus leading to robust induction of cytokine expression. Taken together, our findings identify TLR10 as an important innate immune sensor of viral infection and its role in innate immune defense and immunopathology following viral and bacterial pathogens deserves attention.

Antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus.

BACKGROUND: Public health risks associated to infection by human coronaviruses remain considerable and vaccination is a key option for preventing the resurgence of severe acute respiratory syndrome coronavirus (SARS-CoV). We have previously reported that antibodies elicited by a SARS-CoV vaccine candidate based on recombinant, full-length SARS-CoV Spike-protein trimers, trigger infection of immune cell lines. These observations prompted us to investigate the molecular mechanisms and responses to antibody-mediated infection in human macrophages. METHODS: We have used primary human immune cells to evaluate their susceptibility to infection by SARS-CoV in the presence of anti-Spike antibodies. Fluorescence microscopy and real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) were utilized to assess occurrence and consequences of infection. To gain insight into the underlying molecular mechanism, we performed mutational analysis with a series of truncated and chimeric constructs of fragment crystallizable γ receptors (FcγR), which bind antibody-coated pathogens. RESULTS: We show here that anti-Spike immune serum increased infection of human monocyte-derived macrophages by replication-competent SARS-CoV as well as Spike-pseudotyped lentiviral particles (SARS-CoVpp). Macrophages infected with SARS-CoV, however, did not support productive replication of the virus. Purified anti-viral IgGs, but not other soluble factor(s) from heat-inactivated mouse immune serum, were sufficient to enhance infection. Antibody-mediated infection was dependent on signaling-competent members of the human FcγRII family, which were shown to confer susceptibility to otherwise naïve ST486 cells, as binding of immune complexes to cell surface FcγRII was necessary but not sufficient to trigger antibody-dependent enhancement (ADE) of infection. Furthermore, only FcγRII with intact cytoplasmic signaling domains were competent to sustain ADE of SARS-CoVpp infection, thus providing additional information on the role of downstream signaling by FcγRII. CONCLUSIONS: These results demonstrate that human macrophages can be infected by SARS-CoV as a result of IgG-mediated ADE and indicate that this infection route requires signaling pathways activated downstream of binding to FcγRII receptors.

Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a pH- and cysteine protease-independent FcγR pathway.

Public health measures successfully contained outbreaks of the severe acute respiratory syndrome coronavirus (SARS-CoV) infection. However, the precursor of the SARS-CoV remains in its natural bat reservoir, and reemergence of a human-adapted SARS-like coronavirus remains a plausible public health concern. Vaccination is a major strategy for containing resurgence of SARS in humans, and a number of vaccine candidates have been tested in experimental animal models. We previously reported that antibody elicited by a SARS-CoV vaccine candidate based on recombinant full-length Spike-protein trimers potentiated infection of human B cell lines despite eliciting in vivo a neutralizing and protective immune response in rodents. These observations prompted us to investigate the mechanisms underlying antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro. We demonstrate here that anti-Spike immune serum, while inhibiting viral entry in a permissive cell line, potentiated infection of immune cells by SARS-CoV Spike-pseudotyped lentiviral particles, as well as replication-competent SARS coronavirus. Antibody-mediated infection was dependent on Fcγ receptor II but did not use the endosomal/lysosomal pathway utilized by angiotensin I converting enzyme 2 (ACE2), the accepted receptor for SARS-CoV. This suggests that ADE of SARS-CoV utilizes a novel cell entry mechanism into immune cells. Different SARS vaccine candidates elicit sera that differ in their capacity to induce ADE in immune cells despite their comparable potency to neutralize infection in ACE2-bearing cells. Our results suggest a novel mechanism by which SARS-CoV can enter target cells and illustrate the potential pitfalls associated with immunization against it. These findings should prompt further investigations into SARS pathogenesis.

Delta opioid receptors mediate chemotaxis in bone marrow-derived dendritic cells.

Endogenous opioid peptides are locally produced at the inflammatory site where antigens are captured and processed by dendritic cells (DCs). Subsequently, maturing DCs migrate towards draining lymph nodes to initiate T cell response. Given the primordial role of DCs in adaptive immune response, we examined whether opioids may affect the migratory response of DCs. We found that the delta opioid receptor (DOR) mRNA was expressed at low level in bone marrow-derived immature DCs and up-regulated upon DC maturation. Moreover, DOR agonists triggered DC chemotaxis in vitro. In vivo, enkephalins prevented the egress of mature DCs injected into the peritoneal cavity of normal mice. This effect was inhibited by blocking opioid receptors on mature DCs. The cross-talk between CCR7 and DOR receptors that are both up-regulated during DC maturation was then examined. Whereas opioids did not alter the migratory responsiveness to CCR7 ligands, DOR-mediated mobilization of mature DCs was inhibited by CCL19 and CCL21 suggesting that the opioid chemotactic activity decreases as the concentration of the chemokines increases.

Opioid receptor blockade increases the number of lymphocytes without altering T cell response in draining lymph nodes in vivo.

A number of studies have been dedicated to estimate the consequences on immunity of the clinical use of opioids by focusing on mitogen-induced polyclonal proliferation of T cells from blood or spleen. Here we examined, under physiological conditions, the contribution of endogenous opioids in the development of a CD4(+) T cell response within draining lymph nodes. We show in OVA-primed DO11.10 mice that delta-opioid receptors were up-regulated upon T cell activation in vivo and that opioid receptor neutralization increased the number of specific anti-OVA T lymphocytes without promoting their capacity to proliferate. The sensitivity to Fas-mediated apoptosis of T lymphocytes and the synthesis of homeostatic lymphoid chemokines were not either affected suggesting that opioids operate mainly before the entry of T lymphocytes into lymph nodes.

Ezrin interacts with the SARS coronavirus Spike protein and restrains infection at the entry stage.

BACKGROUND: Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process. METHODOLOGY/PRINCIPAL FINDINGS: We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S-pseudotyped particles and potentiated S-dependent membrane fusion. CONCLUSIONS/SIGNIFICANCE: Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.

Opioid receptor blockade reduces Fas-induced hepatitis in mice.

Fas (CD95)-induced hepatocyte apoptosis and cytotoxic activity of neutrophils infiltrating the injured liver are two major events leading to hepatitis. Because it has been reported that opioids, via a direct interaction, sensitize splenocytes to Fas-mediated apoptosis by upregulating Fas messenger RNA (mRNA) and modulated neutrophil activity, we assumed that opioids may participate in the pathophysiology of hepatitis. Using the hepatitis model induced by agonistic anti-Fas antibody in mice, we showed that opioid receptor blockade reduced liver damage and consequently increased the survival rate of animals when the antagonist naltrexone was injected simultaneously or prior to antibody administration. Treatment of mice with morphine enhanced mortality. Naloxone methiodide-a selective peripheral opioid antagonist-had a protective effect, but the absence of opioid receptors in the liver, together with lack of morphine effect in Fas-induced apoptosis of primary cultured hepatocytes, ruled out a direct effect of opioids on hepatocytes. In addition, the neutralization of opioid activity by naltrexone did not modify Fas mRNA expression in the liver as assessed with real-time quantitative polymerase chain reaction. Injured livers were infiltrated by neutrophils, but granulocyte-depleted mice were not protected against the enhancing apoptotic effect of morphine. In conclusion, opioid receptor blockade improves the resistance of mice to Fas-induced hepatitis via a peripheral mechanism that does not involve a down-modulation of Fas mRNA in hepatocytes nor a decrease in proinflammatory activity of neutrophils.

Anti-mu-opioid-receptor IgG antibodies are commonly present in serum from healthy blood donors: evidence for a role in apoptotic immune cell death.

We previously observed the presence of anti-human mu-opioid-receptor (anti-hMOR) autoantibodies in IgG pools prepared from several thousand healthy blood donors. These autoantibodies behaved agonistically because of their ability to bind to the first and third extracellular loops of the receptor. In this study, we found that each healthy donor's serum contained anti-hMOR IgG autoantibodies with a specific activity against both the first and the third extracellular loops of the receptor. Because of the inability of IgG to cross the blood-brain barrier, we investigated the effects of the expression of anti-hMOR autoantibodies on immune cells. In analogy to studies of the effects of morphine, we investigated the ability of antibodies to sensitize splenocytes to Fas (CD95)-mediated apoptosis. We took advantage of the high sequence homology between murine MOR and hMOR extracellular loops to estimate the effect on murine splenocytes of anti-hMOR antibodies raised by immunizing mice. Splenocytes from mice injected with Chinese hamster ovary (CHO) cells expressing MOR were sensitized to Fas-mediated apoptosis, whereas those from mice injected with CHO cells or phosphate-buffered saline were not. Similar sensitization to Fas-mediated apoptosis was observed in splenocytes from mice undergoing passive transfer either with IgG from mice previously immunized against CHO cells expressing MOR or with IgG directed against the first and third extracellular loops of the receptor. Together, our data show that anti-MOR autoantibodies are commonly expressed in healthy humans and could participate in the control of lymphocyte homeostasis by promoting Fas-mediated apoptosis.