Host phospholipase C-γ1 impairs phagocytosis and killing of mycobacteria by J774A.1 murine macrophages

Macrophages represent the first line of defense against invading Mycobacterium tuberculosis (Mtb). In order to enhance intracellular survival, Mtb targets various components of the host signaling pathways to limit macrophage functions. The outcome of Mtb infection depends on various factors derived from both host and pathogen. A detailed understanding of such factors operating during interaction of the pathogen with the host is a prerequisite for designing new approaches for combating mycobacterial infections. This work analyzed the role of host phospholipase C-γ1 (PLC-γ1) in regulating mycobacterial uptake and killing by J774A.1 murine macrophages. Small interfering RNA mediated knockdown of PLC-γ1 increased internalization and reduced the intracellular survival of both Mtb and Mycobacterium smegmatis (MS) by macrophages. Down-regulation of the host PLC-γ1 was observed during the course of mycobacterial infection within these macrophages. Finally, Mtb infection also suppressed the expression of pro-inflammatory cytokine tumor necrosis factor-α and chemokine (C-C motif) ligand 5 (RANTES) which was restored by knocking down PLC-γ1 in J774A.1 cells. These observations suggest a role of host PLC-γ1 in the uptake and killing of mycobacteria by murine macrophages.     

Toxoplasma gondii infection of activated J774-A1 macrophages causes inducible nitric oxide synthase degradation by the proteasome pathway

Classically activated macrophages produce nitric oxide (NO), which is a potent microbicidal agent. NO production is catalyzed by inducible nitric oxide synthase (iNOS), which uses arginine as substrate producing NO and citruline. However, it has been demonstrated that NO production is inhibited after macrophage infection of Toxoplasma gondii, the agent of toxoplasmosis, due to iNOS degradation. Three possible iNOS degradation pathways have been described in activated macrophages: proteasome, calpain and lysosomal. To identify the iNOS degradation pathway after T. gondii infection, J774-A1 macrophage cell line was activated with lipopolysaccharide and interferon-gamma for 24 h, treated with the following inhibitors, lactacystin (proteasome), calpeptin (calpain), or concanamycin A (lysosomal), and infected with the parasite. NO production and iNOS expression were evaluated after 2 and 6 h of infection. iNOS was degraded in J774-A1 macrophages infected with T. gondii. However, treatment with lactacystin maintained iNOS expression in J774-A1 macrophages infected for 2 h by T. gondii, and after 6 h iNOS was localized in aggresomes. iNOS was degraded after parasite infection of J774-A1 macrophages treated with calpeptin or concanamycin A. NO production confirmed iNOS expression profiles. These results indicate that T. gondii infection of J774-A1 macrophages caused iNOS degradation by the proteasome pathway.     

Responses of inbred mouse strains to infection with intestinal nematodes

Comparisons were made of the immune and inflammatory responses of four strains of inbred mice to infection with the intestinal nematodes Trichinella spiralis and Nippostrongylus brasiliensis to determine whether genetically determined 'high responsiveness' to infection, seen most clearly in intestinal responses, is independent of the parasite concerned and necessarily correlated with protection. The time course of infection was followed by counting adult worms at intervals after infection. Mucosal mast cells and Paneth cell numbers were determined as indices of the intestinal inflammatory response. Levels of IgG2a and IgG1 antibodies and of the cytokines IFN-gamma and IL-5 released from in vitro-stimulated mesenteric node lymphocytes were measured to assess type 1 and type 2 responses. NIH and CBA mice were the most resistant to T. spiralis and N. brasiliensis respectively, resistance in each case being correlated with the most intense intestinal inflammatory responses. C57BL/10 (B10) and B10.BR were the least resistant to T. spiralis, but were as resistant as CBA to N. brasiliensis, despite their intestinal inflammatory responses to both parasites being much lower than the other two strains. Mice infected with T. spiralis made the expected switch from a type 1 (IFN-gamma) to a type 2 (IL-5) response between days 2 and 8, and there were no significant differences in levels of these cytokines between the strains. In contrast, when infected with N. brasiliensis, CBA showed an IFN-gamma response at day 4, all strains switching to IL-5 by day 8 and NIH mice releasing the greatest amount of IL-5. The results indicate that the "high responder" phenotype to intestinal nematode infection is in part determined by host characteristics, but is also determined by the parasite concerned--seen most clearly by the differences between NIH and CBA when infected with T. spiralis and N. brasiliensis. The fact that "low responder" B10 background mice were more resistant to N. brasiliensis than "high responder" NIH implies that each parasite elicits a particular pattern of protective host responses, rather than parasites being differentially susceptible to the same response profile.     

15-Deoxy-Δ(12,14)-prostaglandin J(2) attenuates the biological activities of monocyte/macrophage cell lines

Monocytes/macrophages link the innate and adaptive immune systems, and in inflammatory disorders their activation leads to tissue damage. 15-Deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural peroxisome proliferator-activated receptor gamma (PPARγ) ligand, has garnered much interest because it possesses anti-inflammatory properties in a number of experimental models. However, whether it regulates monocytes/macrophage pathophysiology is still unknown. This study was designed to examine the effects of 15d-PGJ(2) on the phagocytosis, proliferation and inflammatory cytokines generation in mouse monocyte/macrophage cell line RAW264.7 and J774A.1 cells upon lipopolysaccharide challenge. Our results showed that 15d-PGJ(2) inhibited the phagocytic activity and cell proliferation in a dose-dependent manner, and suppressed proinflammatory cytokines expression, such as tumor necrosis factor-α, transforming growth factor-β1, interleukin-6, and monocyte chemotactic protein-1. These effects were independent of PPARγ, because PPARγ agonist (troglitazone or ciglitazone) and PPARγ antagonist (GW9662) did not affect these activities mentioned above in cells. Treatment of 15d-PGJ(2) also did not modulate expression and distribution of PPARγ. However, these effects of 15d-PGJ(2) were abrogated by antioxidant N-acetylcysteine. Moreover, treatment of 15d-PGJ(2) induced a significant increase in reactive oxygen species production in RAW264.7 and J774A.1 cells. In conclusion, 15d-PGJ(2) attenuates the biological activities of mouse monocyte/macrophage cell line cells involving oxidative stress, independently of PPARγ. These data further underline the anti-inflammation potential of 15d-PGJ(2).     

Trichinella spiralis shares epitopes with human autoantigens

Like other helminths, Trichinella spiralis has evolved strategies to allow it to survive in the host organism, including the expression of epitopes similar to those present in either expressed or hidden host antigens. To identify T. spiralis-derived antigens that are evolutionarily conserved in the parasite and its host and that could be responsible for its evasion of the host immune response, we examined the reactivity of six different types of autoantibodies to T. spiralis larvae from muscle. T. spiralis antigens that share epitopes with human autoantigens were identified by assessing the cross-reactivity of autoantibody-containing serum samples with T. spiralis antigens in the absence of specific anti-parasite antibodies. Of the 55 autoantibody-containing human serum samples that we analysed by immunohistological screening, 24 (43.6%) recognised T. spiralis muscle larvae structures such as the subcuticular region, the genital primordium or the midgut. Using Western blots, we demonstrated that the same sera reacted with 24 protein components of T. spiralis muscle larvae excretory-secretory L1 antigens. We found that the human autoantibodies predominantly bound antigens belonging to the TSL1 group; more specifically, the autoantibody-containing sera reacted most frequently with the 53-kDa component. Thus, this protein is a good candidate for further studies of the mechanisms of T. spiralis-mediated immunomodulation.     

Interleukin-4 is chemotactic for mouse macrophages.

An important component of the cell-mediated immune response often is the migration of macrophages to the site of immune activity. Although much evidence suggests that macrophage migration is regulated by antigen-specific T cells, the influence of T cell-derived cytokines on macrophage chemotaxis has not been well studied. Here we present evidence that interleukin-4 (IL-4), a cytokine derived from T helper 2 (Th 2) cells, is chemotactic for mouse peritoneal macrophages. In an in vitro chemotaxis assay using Boyden chambers, recombinant IL-4 was chemotactic for mouse peritoneal exudate macrophages. This response was inhibited in a dose-dependent manner by the anti-IL-4 antibody, 11B11. As shown here and previously, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), cytokines derived from T helper 1 cells, are not chemotactic for mouse macrophages.     

Trichinella spiralis infections of inbred mice: genetics of the host response following infection with different Trichinella isolates

The immune response of inbred strains of mice was studied following infection with isolates of Trichinella from a pig (P1), an arctic fox (AF1), and T. spiralis var. pseudospiralis (TP). Strains of mice previously characterized as highly resistant to a separate pig isolate of T. spiralis responded to the P1 and AF1 isolates by expelling over 80% of the worms by day 10 postinfection (PI), and by suppressing the in vitro release of newborn larvae by female worms. However, the response induced by AF1 worms was expressed more quickly when compared to responses induced by the P1 and TP isolates. The host response to TP was less as recovery was always higher at day 10 PI and antifecundity effects were not induced in TP worms even in highly resistant strains of mice. Strains of mice previously characterized as susceptible to T. spiralis infection were slow to develop resistance when compared to the resistant mouse strains, but even among the susceptible strains, infection with AF1 induced a more rapid response. The mouse strains used in these experiments allowed us to assess the role of the major histocompatibility complex (MHC) and/or non-MHC genes in influencing the responses observed. As previously reported for a pig isolate of T. spiralis, both MHC and non-MHC genes influenced the rate at which worms were expelled from the gut and the host response that limits the fecundity of adult female worms.     

Role of macrophages in early host resistance to respiratory Acinetobacter baumannii infection

Acinetobacter baumannii is an emerging bacterial pathogen that causes nosocomial pneumonia and other infections. Although it is recognized as an increasing threat to immunocompromised patients, the mechanism of host defense against A. baumannii infection remains poorly understood. In this study, we examined the potential role of macrophages in host defense against A. baumannii infection using in vitro macrophage culture and the mouse model of intranasal (i.n.) infection. Large numbers of A. baumannii were taken up by alveolar macrophages in vivo as early as 4 h after i.n. inoculation. By 24 h, the infection induced significant recruitment and activation (enhanced expression of CD80, CD86 and MHC-II) of macrophages into bronchoalveolar spaces. In vitro cell culture studies showed that A. baumannii were phagocytosed by J774A.1 (J774) macrophage-like cells within 10 minutes of co-incubation, and this uptake was microfilament- and microtubule-dependent. Moreover, the viability of phagocytosed bacteria dropped significantly between 24 and 48 h after co-incubation. Infection of J774 cells by A. baumannii resulted in the production of large amounts of proinflammatory cytokines and chemokines, and moderate amounts of nitric oxide (NO). Prior treatment of J774 cells with NO inhibitors significantly suppressed their bactericidal efficacy (P<0.05). Most importantly, in vivo depletion of alveolar macrophages significantly enhanced the susceptibility of mice to i.n. A. baumannii challenge (P<0.01). These results indicate that macrophages may play an important role in early host defense against A. baumannii infection through the efficient phagocytosis and killing of A. baumannii to limit initial pathogen replication and the secretion of proinflammatory cytokines and chemokines for the rapid recruitment of other innate immune cells such as neutrophils.     

IL-10 induces gene expression in macrophages: partial overlap with IL-5 but not with IL-4 induced genes

The hypothesis that IL-10, in addition to down-regulating pro-inflammatory activities of macrophages, induces an alternative state of macrophage reactivity was tested. We therefore conducted a systematic search for genes induced by IL-10 using the method of suppression subtractive hybridisation. Of an initial 1,300 candidate clones obtained, several screening rounds led to the identification of 51 clones which were reproducibly at least twofold up-regulated in mouse J774 macrophages in response to treatment with IL-10. Of these, 41 genes were homologous to known genes involved in cell metabolism or immunoregulation, five contained novel sequences and another five were homologous to ESTs without known function. One major finding was that about 25% of the IL-10 genes were also found expressed in response to IFNgamma, and several of these also reappeared in IL-4 or IL-5 induced mRNA species. Hence, Th1 and Th2 type cytokines may elicit a common basal activation response in macrophages. The second major finding was that 57% of IL-10 induced genes reappeared in IL-5 induced mRNA but no more than 18% were also found in IL-4 induced mRNA of J774 cells. We conclude that the gene expression response to IL-10 in macrophages is partially different from the response to IL-5 and is substantially different from the response to IL-4, which suggests an unexpected diversity of biological phenotypes induced by different Th2 type cytokines.     

Modulation of macrophage cytokine production by ES-62, a secreted product of the filarial nematode Acanthocheilonema viteae.

Parasite survival and host health may depend on the ability of the parasite to modulate the host immune response by the release of immunomodulatory molecules. Excretory-secretory (ES)-62, one such well-defined molecule, is a major secreted protein of the rodent filarial nematode Acanthocheilonema viteae, and has homologues in human filarial nematodes. Previously we have shown that ES-62 is exclusively associated with a Th2 Ab response in mice. Here we provide a rationale for this polarized immune response by showing that the parasite molecule suppresses the IFN-gamma/LPS-induced production, by macrophages, of bioactive IL-12 (p70), a key cytokine in the development of Th1 responses. This suppression of the induction of a component of the host immune response extends to the production of the proinflammatory cytokines IL-6 and TNF-alpha, but not NO. The molecular mechanism underlying these findings awaits elucidation but, intriguingly, the initial response of macrophages to ES-62 is to demonstrate a low and transient release of these cytokines before becoming refractory to further release induced by IFN-gamma/LPS. The relevance of our observations is underscored by the finding that macrophages recovered from mice exposed to "physiological" levels of ES-62 by the novel approach of continuous release from implanted osmotic pumps in vivo were similarly refractory to release of IL-12, TNF-alpha, IL-6, but not NO, ex vivo. Therefore, our results suggest that exposure to ES-62 renders macrophages subsequently unable to produce Th1/proinflammatory cytokines. This likely contributes to the generation of immune responses with an anti-inflammatory Th2 phenotype, a well-documented feature of filarial nematode infection.     

Discoidin domain receptor 1 mediates collagen-induced nitric oxide production in J774A.1 murine macrophages

Nitric oxide (NO) is an important regulator of immune responses. Effects of cytokines, such as tumor necrosis factor (TNF)-alpha or IFN-gamma, and bacterial products, such as lipopolysaccharide, on macrophage NO production have been well documented; however, the role of the extracellular matrix proteins, including collagen, in this process remains unclear. We previously reported that discoidin domain receptor 1 (DDR1), a nonintegrin collagen receptor, was expressed in human macrophages, and its activation facilitated their differentiation as well as cytokine/chemokine production. Here, we examined the role for DDR1 in collagen-induced NO production using the murine macrophage cell line J774 cells that endogenously express DDR1. Activation of J774 cells with collagen induced the expression of inducible NO synthase (iNOS) and NO production. Inhibition of DDR1, but not beta1-integrins, abolished collagen-induced iNOS and NO production. Activation of J774 cells with collagen-activated nuclear factor-kappaB, p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK) and a pharmacological inhibitor of each signaling molecule significantly reduced collagen-induced NO production. Thus, we have demonstrated, for the first time, that the interaction of DDR1 with collagen induces iNOS expression and subsequent NO synthesis in J774 cells through activation of NF-kappaB, p38 MAPK, and JNK and suggest that intervention of DDR1 signaling in macrophages may be useful in controlling inflammatory diseases in which NO plays a critical role.     

CD4+ T Cell-derived IL-10 Promotes Brucella abortus Persistence via Modulation of Macrophage Function

Evasion of host immune responses is a prerequisite for chronic bacterial diseases; however, the underlying mechanisms are not fully understood. Here, we show that the persistent intracellular pathogen Brucella abortus prevents immune activation of macrophages by inducing CD4⁺CD25⁺ T cells to produce the anti-inflammatory cytokine interleukin-10 (IL-10) early during infection. IL-10 receptor (IL-10R) blockage in macrophages resulted in significantly higher NF-kB activation as well as decreased bacterial intracellular survival associated with an inability of B. abortus to escape the late endosome compartment in vitro. Moreover, either a lack of IL-10 production by T cells or a lack of macrophage responsiveness to this cytokine resulted in an increased ability of mice to control B. abortus infection, while inducing elevated production of pro-inflammatory cytokines, which led to severe pathology in liver and spleen of infected mice. Collectively, our results suggest that early IL-10 production by CD25⁺CD4⁺ T cells modulates macrophage function and contributes to an initial balance between pro-inflammatory and anti-inflammatory cytokines that is beneficial to the pathogen, thereby promoting enhanced bacterial survival and persistent infection.     

Isolation of infective promastigotes of Leishmania major from long-term culture by cocultivation with macrophage cell line.

Research on Leishmania-macrophage interaction is mainly focused on the impact of the parasite on macrophages and several known virulent factors have been described. Furthermore, studies on macrophage revealed several defense mechanisms including various cytokines which are released by macrophages to defend against parasite. In the present study, a new aspect of this interaction was evaluated: parasite characteristics, which emerge when they were cocultivated with macrophage. Two promastigote characteristics, survival at high temperature (32 degrees C) and infectivity rate were the focus of this study. In this study, an in vitro coculture model for promastigotes with macrophage cell line, J774 A1, was introduced using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane. After 5-7 days of coculturing at 32 degrees C, a few promastigotes survived longer than control group. Once this population of parasite was cultured at optimal temperature (26 degrees C), the emerged new clone was much more infective for J774 A1 cell line in comparison with the original one. Having this system and using the new clone of promastigotes, parasite infectivity rate was raised from 1-2% of original clone to 35-45%. Using this new introduced technique, infective promastigotes were isolated from 9 month old frequently sub-cultured clone of Leishmania major. This coculturing system allows investigators to prepare infective promastigotes from the frequently cultured parasites. Molecular and biochemical mechanisms of this phenomenon need to be investigated.     

Acylation determines the toll-like receptor (TLR)-dependent positive versus TLR2-, mannose receptor-, and SIGNR1-independent negative regulation of pro-inflammatory cytokines by mycobacterial lipomannan

Mycobacterium tuberculosis lipomannans (LMs) modulate the host innate immune response. The total fraction of Mycobacterium bovis BCG LM was shown both to induce macrophage activation and pro-inflammatory cytokines through Toll-like receptor 2 (TLR2) and to inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages through a TLR2-independent pathway. The pro-inflammatory activity was attributed to tri- and tetra-acylated forms of BCG LM but not the mono- and di-acylated ones. Here, we further characterize the negative activities of M. bovis BCG LM on primary murine macrophage activation. We show that di-acylated LMs exhibit a potent inhibitory effect on cytokine and NO secretion by LPS-activated macrophages. The inhibitory activity of mycobacterial mannose-capped lipoarabino-mannans on human phagocytes was previously attributed to their binding to the C-type lectins mannose receptor or specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). However, we found that di-acylated LM inhibition of LPS-induced tumor necrosis factor secretion by murine macrophages was independent of TLR2, mannose receptor, or the murine ortholog SIGNR1. We further determined that tri-acyl-LM, an agonist of TLR2/TLR1, promoted interleukin-12 p40 and NO secretion through the adaptor proteins MyD88 and TIRAP, whereas the fraction containing tetra-acylated LM activated macrophages in a MyD88-dependent fashion, mostly through TLR4. TLR4-dependent pro-inflammatory activity was also seen with M. tuberculosis LM, composed mostly of tri-acylated LM, suggesting that acylation degree per se might not be sufficient to determine TLR2 versus TLR4 usage. Therefore, LM acylation pattern determines the anti-inflammatory versus pro-inflammatory effects of LM through different pattern recognition receptors or signaling pathways and may represent an additional mean of regulating the host innate immunity by mycobacteria.     

Macrophage migration inhibitory factor exhibits a pronounced circadian rhythm relevant to its role as a glucocorticoid counter-regulator

In humans, maximal expression of T helper 1 cytokines coincide with the nocturnal nadir of plasma cortisol, whereas T helper 2 cytokine responses are dominant during day-time. The pro-inflammatory cytokine, macrophage migration inhibitory factor counter-regulates glucocorticoid-mediated immune suppression. To determine the relationship between cortisol and macrophage migration inhibitory factor, healthy volunteers had blood drawn hourly for 24 h for measurement of plasma cortisol and basal- and stimulated-macrophage migration inhibitory factor. Similar to cortisol, macrophage migration inhibitory factor peaked during the late morning whereas interferon-gamma, tumour necrosis factor-alpha, interleukin-1 and interleukin-12 demonstrated a nocturnal peak. After oral cortisone, plasma macrophage migration inhibitory factor rose 2-4-fold, whereas the other cytokines decreased. There was no correlation between cortisol during the insulin tolerance test and plasma macrophage migration inhibitory factor. The late morning peak of macrophage migration inhibitory factor, by antagonizing cortisol-mediated pro-inflammatory cytokine suppression may prolong the duration of early morning inflammation. These observations explain the beneficial role of macrophage migration inhibitory factor neutralization in models of inflammatory arthritis.     

Human recombinant IL-10 reduces xenogenic cytotoxicity via macrophage M2 polarization

Xenotransplantation has been considered an alternative to the moderate shortage of donor organs for transplantation. To achieve successful xenotransplatation, there is the need to overcome immune rejection. Although, hyperacute rejection has been overcome by α1,3-galactosyltransferase knockout pig, cellular immune rejection remains as a subsequent barrier. Interleukin-10 (IL-10) is known as an anti-inflammatory and immunomodulatory cytokine which has been shown to limit inflammatory responses by inhibiting macrophage activation in several animal experiments. To study the effect of human IL-10 (hIL-10) on pig-to-human xenotransplantation, porcine kidney epithelial cell line (PK(15)) expressing hIL-10 was established. The cytotoxicity of macrophages decreased by hIL-10 from transgenic cells. Furthermore, there is a decreased production of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-23, and increased anti-inflammatory cytokines like IL-10, but not transforming growth factor beta, in the presence of hIL-10. Also, macrophage polarization toward M2-like phenotype were induced by hIL-10 from transgenic PK(15) cells. Finally, we suggest that the cytotoxicity of human macrophages was reduced by hIL-10 from transgenic cells, inducing M2-like macrophage polarization. Therefore, these results show that hIL-10 transgenic pig can be used as a model to overcome acute immune rejection in pig-to-human xenotransplantation.     

Isolation of infective promastigotes of Leishmania major from long-term culture by cocultivation with macrophage cell line

Research on Leishmania–macrophage interaction is mainly focused on the impact of the parasite on macrophages and several known virulent factors have been described. Furthermore, studies on macrophage revealed several defense mechanisms including various cytokines which are released by macrophages to defend against parasite. In the present study, a new aspect of this interaction was evaluated: parasite characteristics, which emerge when they were cocultivated with macrophage. Two promastigote characteristics, survival at high temperature (32 °C) and infectivity rate were the focus of this study. In this study, an in vitro coculture model for promastigotes with macrophage cell line, J774 A1, was introduced using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane. After 5–7 days of coculturing at 32 °C, a few promastigotes survived longer than control group. Once this population of parasite was cultured at optimal temperature (26 °C), the emerged new clone was much more infective for J774 A1 cell line in comparison with the original one. Having this system and using the new clone of promastigotes, parasite infectivity rate was raised from 1–2% of original clone to 35–45%. Using this new introduced technique, infective promastigotes were isolated from 9 month old frequently sub-cultured clone of Leishmania major. This coculturing system allows investigators to prepare infective promastigotes from the frequently cultured parasites. Molecular and biochemical mechanisms of this phenomenon need to be investigated.