Gamma interferon is not essential in host defense against disseminated candidiasis in mice.

In vitro studies have suggested a role for interferon gamma (IFN-gamma) in host defense against disseminated candidiasis, but in vivo studies are inconclusive. We utilized homozygous IFN-gamma knockout (GKO) mice to determine if the cytokine is essential in host defense against this disease. Genotypes of mice were determined by PCR with specific primers for the normal or disrupted IFN-gamma gene. The GKO status of the mice was confirmed by an enzyme-linked immunosorbent assay, which showed no detectable IFN-gamma produced by their splenocytes stimulated by concanavalin A. To test the susceptibility of GKO mice to candidiasis, the animals were infected either intravenously (i.v.) or intragastrically (i.g.) with Candida albicans. GKO mice infected i.v. survived as long as wild-type (WT) mice and showed no difference in Candida CFU counts in liver, spleen, or kidneys compared to those for WT mice. When animals were given Candida i.g., at 3 h or at 10 or 21 days after infection, there was no dissemination of Candida to the lung, liver, spleen, or kidneys for either GKO or WT mice. There was no difference in Candida CFU counts recovered from the stomach or intestines between GKO and WT mice. Histological examination of the stomach cardial-atrium fold, where the fungus was located, showed that GKO mice did not have evidence of more tissue damage or fungal invasion than WT mice. Finally, the jejunum for both types of mice showed no evidence of tissue damage or fungal invasion. These studies indicate that IFN-gamma is not essential in host defense against C. albicans that originates from a mucosal site or that is given directly into the bloodstream in a mouse model.     

Immune protection against septic peritonitis in endotoxin-primed mice is related to reduced neutrophil apoptosis

The innate immune system provides essential information about the presence of infectious danger and signals the activation and instruction of adaptive immunity. The present study addressed the question of whether prior exposure of the innate immune system to LPS may modulate host defense against acute septic peritonitis. We show that LPS priming 4 days, but not 2 days, prior to infection enhances bacterial clearance and improves survival of septic peritonitis. Immune protection in day 4 LPS-primed mice was specifically associated with a marked increase in the accumulation and activation of neutrophils at the site of infection. Accumulating neutrophils in day 4 LPS-primed mice exhibited a normal production of reactive oxygen metabolites in response to in vivo exposure to intestinal bacteria. The local increase in neutrophil numbers was found to result from a reduced rate of apoptotic cell death. Inhibition of neutrophil apoptosis in LPS-primed mice was mediated by soluble factor(s) distinct from G-CSF and GM-CSF. Thus, engagement of pattern recognition systems prior to infection may improve host defense by amplifying the effector cell response of innate immunity. The results also provide in vivo evidence that apoptosis of inflammatory cells represents an important process for the control of host defense to infection.     

MyD88 and TLR2, but not TLR4, are required for host defense against Cryptococcus neoformans

We investigated here the potential role of Toll-like receptors (TLR) and the adaptor protein MyD88 in innate immunity responses to Cryptococcus neoformans, a pathogenic encapsulated yeast. Peritoneal macrophages from MyD88(-/-) or TLR2(-/-) mice released significantly less TNF-alpha, compared with wild-type controls, after in vitro stimulation with whole yeasts. In contrast, no differences in TNF-alpha release were noted between macrophages from C3H/HeJ mice, which have a loss of function mutation in TLR4, relative to C3H/HeN controls. When MyD88- or TLR2-deficient mice were infected with low doses of the H99 serotype A strain, all of the control animals, but none of MyD88(-/-) and only 38% of the TLR2(-/-) animals survived, in association with higher fungal burden in the mutant mice. Both MyD88(-/-) and TLR2(-/-) animals showed decreased TNF-alpha, IL-12p40 and/or IFN-gamma expression in various organs during infection. No difference in susceptibility to experimental cryptococcosis was found between C3H/HeJ mice and C3H/HeN controls. In conclusion, our data indicate that TLR2 and MyD88, but not TLR4, critically contribute to anti-cryptococcal defenses through the induction of increased TNF-alpha, IL-12 and IFN-gamma expression.     

Nonredundant roles of TIRAP and MyD88 in airway response to endotoxin, independent of TRIF, IL-1 and IL-18 pathways

Inhaled endotoxins induce an acute inflammatory response in the airways mediated through Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). However, the relative roles of the TLR4 adaptor proteins TIRAP and TRIF and of the MyD88-dependent IL-1 and IL-18 receptor pathways in this response are unclear. Here, we demonstrate that endotoxin-induced acute bronchoconstriction, vascular damage resulting in protein leak, Th1 cytokine and chemokine secretion and neutrophil recruitment in the airways are abrogated in mice deficient for either TIRAP or MyD88, but not in TRIF deficient mice. The contribution of other TLR-independent, MyD88-dependent signaling pathways was investigated in IL-1R1, IL-18R and caspase-1 (ICE)-deficient mice, which displayed normal airway responses to endotoxin. In conclusion, the TLR4-mediated, bronchoconstriction and acute inflammatory lung pathology to inhaled endotoxin critically depend on the expression of both adaptor proteins, TIRAP and MyD88, suggesting cooperative roles, while TRIF, IL-1R1, IL-18R signaling pathways are dispensable.     

Gamma interferon as a crucial host defense against Rickettsia conorii in vivo.

Gamma interferon (IFN-gamma) plays an important role as a host defense in rickettsial infection. Swiss Webster mice, which are resistant to Rickettsia conorii (Malish 7 strain) infection, were treated with a monoclonal antibody against mouse IFN-gamma. When the antibody-treated mice were inoculated with 12 50% tissue culture infective doses of R. conorii, the mortality was 47% and the morbidity was 100%. None of the control mice, which received the same dose of R. conorii, died or became ill. The enumeration of rickettsiae in organs by direct immunofluorescence in paraffin sections demonstrated higher quantities of rickettsiae in the spleen had liver of IFN-gamma-depleted mice as compared with those of the infected controls. The kinetic analysis of IFN-gamma levels in sera showed depletion in the treated mice. These results indicate that IFN-gamma plays an important role as a host defense in the early stage of rickettsial infection. Survival of some mice despite continued treatment with antibody to IFN-gamma suggests that other immune mechanisms may also be important.     

Toll-like receptor signal adaptor protein MyD88 is required for sustained endotoxin-induced acute hypoferremic response in mice

Hypoferremia, associated with immune system activation, involves a marked reduction in the levels of circulating iron, coupled with iron sequestration within macrophages. Toll-like receptor (TLR) signaling plays an important role in the development of the hypoferremic response, but how downstream signaling events affect genes involved in iron metabolism is incompletely understood. We investigated the involvement of MyD88-dependent (MyD88) and MyD88-independent (TRIF) TLR signaling in the development of hypoferremia. Using MyD88-deficient and TRIF-deficient mice, we show that MyD88 and TRIF signaling pathways are critical for up-regulation by lipopolysaccharide (LPS) of the iron regulator hepcidin. In addition, MyD88 signaling is required for the induction of lipocalin 2 secretion and iron sequestration in the spleen. Activation of TLR4 and TLR3 signaling through LPS and polyinosinic:polycytidylic acid [poly(I:C)] treatments resulted in rapid down-regulation of HFE protein [encoded by the hemochromatosis gene (Hfe)] and ferroportin [encoded by solute carrier family 40 (iron-regulated transporter), member 1 (Slc40a1)] expression in the spleen, independent of MyD88 or TRIF signaling and proinflammatory cytokine production. However, lack of MyD88 signaling significantly impaired the hypoferremic response triggered by LPS, indicating that ferroportin and HFE protein down-regulation alone are insufficient to maintain hypoferremia. The extent of the hypoferremic response was found to be limited by initial, basal iron levels. Together, these results suggest that targeting specific TLR signaling pathways by affecting the function of adaptor molecules may provide new strategies to counteract iron sequestration within macrophages during inflammation.     

Chronic pneumonia despite adaptive immune response to Mycobacterium bovis BCG in MyD88-deficient mice

To assess the role of Toll-like receptor (TLR) signalling in host response to mycobacterial infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) were infected with the vaccine strain Mycobacterium bovis (BCG), and the immune response and bacterial burden were investigated. Macrophages and dendritic cells from MyD88-deficient mice stimulated in vitro with BCG mycobacterial antigens produced very low levels of proinflammatory cytokines, while the expression of costimulatory molecules such as CD40 and CD86 was preserved. Upon systemic infection with BCG (2 x 10(6) CFU i.v.) MyD88-deficient mice developed confluent chronic pneumonia with two log higher CFU than wild-type mice. Interestingly, the infection was controlled in liver and spleen and there was efficient systemic T-cell priming with high IFNgamma production by CD4+ splenic T cells in MyD88-deficient mice. Lung infiltrating cells showed IFNgamma production by pulmonary CD4+ T cells upon specific restimulation, and a reduced capacity to produce nitric oxide and IL-10. In summary, despite the dramatic reduction of the innate immune response, MyD88-deficient mice were able to mount an efficient T-cell response to mycobacterial antigens, which was however insufficient to control infection in the lung, resulting in chronic pneumonia in MyD88-deficient mice.     

Restoration by recombinant interferon alpha A/D of host defense systems against tumor in immunosuppressed mice

Recombinant human interferon alpha A/D (A/D) restored or augmented host defense systems against tumors in immunosuppressed mice. In normal C57BL/6 mice, inoculation of B16 melanoma F1 cells caused few pulmonary metastasis, whereas in mice pretreated with cyclophosphamide (CY) it caused a high incidence of pulmonary metastasis, leading to earlier death than in the normal mice inoculated with the same dose of the tumor. A/D given after the CY treatment counteracted the deleterious effects of the CY treatment. Since such restorative activity was seen even against the subline of B16 F1 which had been made resistant to its direct antiproliferative effect, A/D seems to exert its effect indirectly through host defense systems. However, this activity of A/D in the mice pretreated with CY was abrogated by inoculation of anti-asialo GM₁ serum but not by i-carrageenan. The CY treatment reduced NK activity, while A/D given after the CY treatment restored or augmented the NK cell activity in lung cells and peripheral blood mononuclear cells, but not in spleen cells. These findings suggest that the restoration or augmentation of NK activity in the lung and/or peripheral blood might be the major factor leading to the antimetastatic activity of A/D in the mice treated with CY.     

Improved antibacterial host defense and altered peripheral granulocyte homeostasis in mice lacking the adhesion class G protein receptor CD97

CD97 is a member of the adhesion family of G protein-coupled receptors. Alternatively spliced forms of CD97 bind integrins alpha5beta1 and alphavbeta3, decay accelerating factor, or dermatan sulfate. CD97 is expressed on myeloid cells at high levels and a variety of other cell types at lower levels. Little is known about the physiological function of CD97. To begin dissecting the function of CD97, we evaluated the immune response of CD97 null mice to systemic infection by Listeria monocytogenes. CD97 null mice were significantly more resistant to listeriosis than matched wild-type mice. A major determinant of the difference in survival appeared to be the comparatively more robust accumulation of granulocytes in the blood and in infected livers of CD97 null mice within 18 h of inoculation, correlating with a decrease in the number of bacteria. CD97 null mice also displayed a mild granulocytosis in the nonchallenged state. Because there is a strong suggestion that CD97 functions in an adhesive capacity, we examined the migratory properties of granulocytes in CD97 null mice. In chimeric animals, CD97 null and wild-type granulocytes migrated similarly, as determined by inflammation-induced emigration from the bone marrow and accumulation in the peritoneum. Granulocyte development in the bone marrow of CD97 null mice was comparable to that of wild-type mice, and CD97 deficiency did not appear to stimulate granulocytosis secondary to peripheral inflammation and resultant granulocyte colony-stimulating factor induction, unlike various other models of adhesion deficiencies. Our results suggest that CD97 plays a role in peripheral granulocyte homeostasis.     

MyD88 and TRIF synergistic interaction is required for TH1‐cell polarization with a synthetic TLR4 agonist adjuvant

Glucopyranosyl lipid adjuvant‐stable emulsion (GLA‐SE) is a synthetic adjuvant TLR4 agonist that promotes potent poly‐functional T_H1 responses. Different TLR4 agonists may preferentially signal via MyD88 or TIR‐domain‐containing adapter inducing IFN‐beta (TRIF) to exert adjuvant effects; however, the contribution of MyD88 and TRIF signaling to the induction of polyclonal T_H1 responses by TLR4 agonist adjuvants has not been studied in vivo. To determine whether GLA‐SE preferentially signals through MyD88 or TRIF, we evaluated the immune response against a candidate tuberculosis (TB) vaccine Ag following immunization of mice lacking either signaling adapter compared with that of wild‐type mice. We find that both MyD88 and TRIF are necessary for GLA‐SE to induce a poly‐functional T_H1 immune response characterized by CD4⁺ T cells producing IFN‐γ, TNF, and IL‐2, as well as IgG2c class switching, when paired with the TB vaccine Ag ID93. Accordingly, the protective efficacy of ID93/GLA‐SE immunization against aerosolized Mycobacterium tuberculosis was lost when either signaling molecule was ablated. We demonstrate that MyD88 and TRIF must be expressed in the same cell for the in vivo T_H1‐skewing adjuvant activity, indicating that these two signaling pathways cooperate on an intracellular level. Thus engagement of both the MyD88 and TRIF signaling pathways are essential for the effective adjuvant activity of this TLR4 agonist.     

Wound healing is impaired in MyD88-deficient mice: a role for MyD88 in the regulation of wound healing by adenosine A2A receptors

Synergy between Toll-like receptor (TLR) and adenosine A2A receptor (A2AR) signaling switches macrophages from production of inflammatory cytokines such as tumor necrosis factor-alpha to production of the angiogenic growth factor vascular endothelial growth factor (VEGF). We show in this study that this switch critically requires signaling through MyD88, IRAK4, and TRAF6. Macrophages from mice lacking MyD88 (MyD88(-/-)) or IRAK4 (IRAK4(-/-)) lacked responsiveness to TLR agonists and did not respond to A2AR agonists by expressing VEGF. Suppression of TRAF6 expression with siRNA in RAW264.7 macrophages also blocked their response to TLR and A2AR agonists. Excisional skin wounds in MyD88(-/-) mice healed at a markedly slower rate than wounds in wild-type MyD88(+/+) mice, showing delayed contraction, decreased and delayed granulation tissue formation, and reduced new blood vessel density. Although macrophages accumulated to higher levels in MyD88(-/-) wounds than in controls, expression of VEGF and HIF1-alpha mRNAs was elevated in MyD88(+/+) wounds. CGS21680, an A2AR agonist, promoted repair in MyD88(+/+) wounds and stimulated angiogenesis but had no significant effect on healing of MyD88(-/-) wounds. These results suggest that the synergistic interaction between TLR and A(2A)R signaling observed in vitro that switches macrophages from an inflammatory to an angiogenic phenotype also plays a role in wound healing in vivo.     

The myeloid differentiation factor 88 is dispensable for the development of a delayed host response to Pseudomonas aeruginosa lung infection in mice

Because MyD88 transduces a core set of Toll-like receptor (TLR)-induced signals, microbial-induced host responses can be divided broadly into the MyD88-dependent and MyD88-independent pathways. A specific pathogen induces a distinct pattern of host response dependent upon the signalling pathways employed. Recently, we demonstrated that a MyD88-dependent pathway is essential for the development of early (4-8 h) host response to Pseudomonas aeruginosa lung infection. Here, we show that the development of a delayed (24-48 h) host response to P. aeruginosa is independent of MyD88. Using MyD88-deficient mice, the production of macrophage inflammatory protein 2, tumour necrosis factor and interleukin 1alpha in the airway was observed following P. aeruginosa lung infection for 24 or 48 h. Moreover, the MyD88-deficient mice recruited sufficient neutrophils in the lung and cleared the bacteria efficiently from the lung after 48 h. Thus, the full development of host responses to P. aeruginosa lung infection involves, in a sequential, stepwise fashion, a MyD88-dependent early response and a MyD88-independent delayed mechanism.     

Staphylococcal enterotoxin A induction of pro-inflammatory cytokines and lethality in mice is primarily dependent on MyD88

Toll‐like receptors (TLRs) are germline‐encoded innate immune receptors that recognize invading micro‐organisms and induce immune and inflammatory responses. Deregulation of TLRs is known to be closely linked to various immune disorders and inflammatory diseases. Cells at sites of inflammation are exposed to hypoxic stress, which further aggravates inflammatory processes. We have examined if hypoxic stress modulates the TLR activity of macrophages. Hypoxia and CoCl₂ (a hypoxia mimetic) enhanced the expression of TLR4 messenger RNA and protein in macrophages (RAW264.7 cells), whereas the messenger RNA of other TLRs was not increased. To determine the underlying mechanism, we investigated the role of hypoxia‐inducible factor 1 (HIF‐1) in the regulation of TLR4 expression. Knockdown of HIF‐1α expression by small interfering RNA inhibited hypoxia‐induced and CoCl₂‐induced TLR4 expression in macrophages, while over‐expression of HIF‐1α potentiated TLR4 expression. Chromatin immunoprecipitation assays revealed that HIF‐1α binds to the TLR4 promoter region under hypoxic conditions. In addition, deletion or mutation of a putative HIF‐1‐binding motif in the TLR4 promoter greatly attenuated HIF‐1α‐induced TLR4 promoter reporter expression. Up‐regulation of TLR4 expression by hypoxic stress enhanced the response of macrophages to lipopolysaccharide, resulting in increased expression of cyclooxygenase‐2, interleukin‐6, regulated on activation normal T cell expressed and secreted, and interferon‐inducible protein‐10. These results demonstrate that TLR4 expression in macrophages is up‐regulated via HIF‐1 in response to hypoxic stress, suggesting that hypoxic stress at sites of inflammation enhances susceptibility to subsequent infection and inflammatory signals by up‐regulating TLR4.     

Role of gamma interferon and interleukin-4 in host defense against the human filarial parasite Brugia malayi

We have investigated the roles of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) in host defense against Brugia malayi. Our data suggest that the lack of either IFN-gamma or IL-4 prolongs the time required to achieve sterile immunity, suggesting that both canonical type 1 and type 2 responses are involved in the clearance of infection.     

Chlamydia muridarum infection elicits a beta interferon response in murine oviduct epithelial cells dependent on interferon regulatory factor 3 and TRIF

Chlamydia trachomatis is the most common sexually transmitted bacterial infection in the United States. Utilizing cloned murine oviduct epithelial cell lines, we previously identified Toll-like receptor 2 (TLR2) as the principal epithelial pattern recognition receptor (PRR) for infection-triggered release of the acute inflammatory cytokines interleukin-6 and granulocyte-macrophage colony-stimulating factor. The infected oviduct epithelial cell lines also secreted the immunomodulatory cytokine beta interferon (IFN-beta) in a largely MyD88-independent manner. Although TLR3 was the only IFN-beta production-capable TLR expressed by the oviduct cell lines, we were not able to determine whether TLR3 was responsible for IFN-beta production because the epithelial cells were unresponsive to the TLR3 ligand poly(I-C), and small interfering RNA (siRNA) techniques were ineffective at knocking down TLR3 expression. To further investigate the potential role of TLR3 in the infected epithelial cell secretion of IFN-beta, we examined the roles of its downstream signaling molecules TRIF and IFN regulatory factor 3 (IRF-3) using a dominant-negative TRIF molecule and siRNA specific for TRIF and IRF-3. Antagonism of either IRF-3 or TRIF signaling significantly decreased IFN-beta production. These data implicate TLR3, or an unknown PRR utilizing TRIF, as the source of IFN-beta production by Chlamydia-infected oviduct epithelial cells.     

Expression of genes encoding innate host defense molecules in normal human monocytes in response to Candida albicans

Little is known about the regulation and coordinated expression of genes involved in the innate host response to Candida albicans. We therefore examined the kinetic profile of gene expression of innate host defense molecules in normal human monocytes infected with C. albicans using microarray technology. Freshly isolated peripheral blood monocytes from five healthy donors were incubated with C. albicans for 0 to 18 h in parallel with time-matched uninfected control cells. RNA from monocytes was extracted and amplified for microarray analysis, using a 42,421-gene cDNA chip. Expression of genes encoding proinflammatory cytokines, including tumor necrosis factor alpha, interleukin 1 (IL-1), IL-6, and leukemia inhibitory factor, was markedly enhanced during the first 6 h and coincided with an increase in phagocytosis. Expression of these genes returned to near baseline by 18 h. Genes encoding chemokines, including IL-8; macrophage inflammatory proteins 1, 3, and 4; and monocyte chemoattractant protein 1, also were strongly up-regulated, with peak expression at 4 to 6 h, as were genes encoding chemokine receptors CCR1, CCR5, CCR7, and CXCR5. Expression of genes whose products may protect monocyte viability, such as BCL2-related protein, metallothioneins, CD71, and SOCS3, was up-regulated at 4 to 6 h and remained elevated throughout the 18-h time course. On the other hand, expression of genes encoding T-cell-regulatory molecules (e.g., IL-12, gamma interferon, and transforming growth factor beta) was not significantly affected during the 18-h incubation. Moreover, genes encoding IL-15, the IL-13 receptor (IL-13Ra1), and CD14 were suppressed during the 18-h exposure to C. albicans. Thus, C. albicans is a potent inducer of a dynamic cascade of expression of genes whose products are related to the recruitment, activation, and protection of neutrophils and monocytes.     

IL-12 and IFN-gamma in host defense against mycobacteria and salmonella in mice and men.

The development of gene-knockout mice and the identification of gene-deficient humans have improved our understanding of the role of IL-12 and IFN-gamma in host defense. Comparison of experimental and natural infections has shown that animals and humans genetically deficient in immunity mediated by IL-12 or IFN-gamma are highly susceptible to mycobacteria and salmonella. Impaired secretion of, or response to, IFN-gamma is the common pathogenic mechanism that accounts for impaired granuloma formation and uncontrolled growth of bacteria within macrophages. The axis formed between IL-12 and IFN-gamma is essential for protective immunity against mycobacteria and salmonella in mice and men.     

Involvement of CD14, toll-like receptors 2 and 4, and MyD88 in the host response to the fungal pathogen Cryptococcus neoformans in vivo

The major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), is recognized by Toll-like receptor 2 (TLR2), TLR4, and CD14. In these studies, mice deficient in CD14, TLR2, TLR4, and the TLR-associated adaptor protein, MyD88, were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses against C. neoformans. MyD88(-/-) mice had significantly reduced survival compared with wild-type C57BL/6 mice after intranasal (i.n.) and intravenous (i.v.) infection with live C. neoformans. CD14(-/-) mice had reduced survival when infected i.v., while TLR2(-/-) mice died significantly earlier after i.n. infection. Mortality was similar comparing TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice following i.v. or i.n. challenge with C. neoformans. The course of pulmonary cryptococcosis was studied in more detail in the CD14(-/-), TLR2(-/-), and MyD88(-/-) mice. MyD88(-/-) mice infected i.n. had higher numbers of CFU in the lungs as well as higher GXM levels in the sera and lungs 7 days after infection than wild-type mice did. Surprisingly, there were no major differences in the levels of tumor necrosis factor alpha, interleukin-4 (IL-4), IL-10, IL-12p70, or gamma interferon in the lungs of C. neoformans-infected knockout mice compared with wild-type mice. Histopathologic analysis of the lungs on day 7 postinfection revealed minimal inflammation in all mouse groups. These studies demonstrate a major role for MyD88 and relatively minor roles for CD14 and TLR2 in the response to cryptococcal infection, with the decreased survival of MyD88(-/-) mice correlating with increased numbers of lung CFU and serum and lung GXM levels.