Christian Capo

The two faces of interleukin 10 in human infectious diseases

Resolution of infections depends on the hosts ability to mount a protective immune response. However, an exacerbated response to infections may result in deleterious lesions. Consequently, immunoregulatory mechanisms are needed to control immune response and prevent infection-associated lesions. Interleukin 10 may be a major regulator of innate and adaptive immunity in vitro and in animals, but its role in human infections is still unclear. Review of the published work reveals wide involvement of interleukin 10 in two major features of infectious diseases. On one hand, interleukin 10 prevents the development of immunopathological lesions that result from exacerbated protective immune response to acute and chronic infections. On the other hand, it is critically involved in persistence of bacteria and viruses by interfering with innate and adaptive protective immunity. Moreover, infections induce the expansion of interleukin-10-producing regulatory cells that are involved in protection against allergic diseases.

Coxiella burnetii Survival in THP-1 Monocytes Involves the Impairment of Phagosome Maturation: IFN-γ Mediates its Restoration and Bacterial Killing

The subversion of microbicidal functions of macrophages by intracellular pathogens is critical for their survival and pathogenicity. The replication of Coxiella burnetii, the agent of Q fever, in acidic phagolysosomes of nonphagocytic cells has been considered as a paradigm of intracellular life of bacteria. We show in this study that C. burnetii survival in THP-1 monocytes was not related to phagosomal pH because bacterial vacuoles were acidic independently of C. burnetii virulence. In contrast, virulent C. burnetii escapes killing in resting THP-1 cells by preventing phagosome maturation. Indeed, C. burnetii vacuoles did not fuse with lysosomes because they were devoid of cathepsin D, and did not accumulate lysosomal trackers; the acquisition of markers of late endosomes and late endosomes-early lysosomes was conserved. In contrast, avirulent variants of C. burnetii were eliminated by monocytes and their vacuoles accumulated late endosomal and lysosomal markers. The fate of virulent C. burnetii in THP-1 monocytes depends on cell activation. Monocyte activation by IFN-γ restored C. burnetii killing and phagosome maturation as assessed by colocalization of C. burnetii with active cathepsin D. In addition, when IFN-γ was added before cell infection, it was able to stimulate C. burnetii killing but it also induced vacuolar alkalinization. These findings suggest that IFN-γ mediates C. burnetii killing via two distinct mechanisms, phagosome maturation, and phagosome alkalinization. Thus, the tuning of vacuole biogenesis is likely a key part of C. burnetii survival and the pathophysiology of Q fever.

Effect of Sex on Coxiella burnetii Infection: Protective Role of 17β-Estradiol

Q fever is a zoonosis caused by Coxiella burnetii and recently has been recognized as a potential agent of bioterrorism. In Q fever, men are symptomatic more often than women, despite equal seroprevalence. We hypothesized that sex hormones play a role in the pathogenesis of C. burnetii infection. When C57/BL6 mice were injected with C. burnetii, bacteria load and granuloma numbers were lower in females than in males. Ovarectomized mice showed increased bacteria load in the spleen and the liver, similar to that found in males. The granuloma number was also increased in ovarectomized mice and reached the levels found in males. Tissue infection and granulomatous response are largely under the control of estrogens: treatment of ovarectomized mice with 17beta-estradiol reduced both bacteria loads and granuloma numbers. These results show that sex hormones control host response to C. burnetii infection and may account for host-dependent clinical presentation of Q fever.

Ameobal pathogen mimivirus infects macrophages through phagocytosis.

Mimivirus, or Acanthamoeba polyphaga mimivirus (APMV), a giant double-stranded DNA virus that grows in amoeba, was identified for the first time in 2003. Entry by phagocytosis within amoeba has been suggested but not demonstrated. We demonstrate here that APMV was internalized by macrophages but not by non-phagocytic cells, leading to productive APMV replication. Clathrin- and caveolin-mediated endocytosis pathways, as well as degradative endosome-mediated endocytosis, were not used by APMV to invade macrophages. Ultrastructural analysis showed that protrusions were formed around the entering virus, suggesting that macropinocytosis or phagocytosis was involved in APMV entry. Reorganization of the actin cytoskeleton and activation of phosphatidylinositol 3-kinases were required for APMV entry. Blocking macropinocytosis and the lack of APMV colocalization with rabankyrin-5 showed that macropinocytosis was not involved in viral entry. Overexpression of a dominant-negative form of dynamin-II, a regulator of phagocytosis, inhibited APMV entry. Altogether, our data demonstrated that APMV enters macrophages through phagocytosis, a new pathway for virus entry in cells. This reinforces the paradigm that intra-amoebal pathogens have the potential to infect macrophages.

Macrophage polarization and bacterial infections.

Purpose of review Macrophages are the first line of defense against pathogens, and the mode of their activation will determine the success or failure of the host response to pathogen aggression. Based on limited numbers of markers, activated macrophages can be classified as classically activated (M1) macrophages that support microbicidal activity or alternatively activated (M2) macrophages that are not competent to eliminate pathogens. The development of high-throughput gene expression methods affords a reappraisal of the concept of macrophage activation in human infectious diseases. Recent findings By combining microarray data and conventional approaches, it is becoming clear that the M1 polarization program is associated with gastrointestinal infections (e.g. typhoid fever and Helicobacter pylori gastritis) and active tuberculosis. An M2 signature is observed in lepromatous leprosy, Whipples disease, and localized infections (keratitis, chronic rhinosinusitis). However, these findings could not be predicted from the analysis of the M1/M2 programs of macrophages stimulated in vitro. Summary The reappraisal of macrophage polarization by high-throughput methods is critical to understanding the role of macrophage polarization in infectious diseases. Only the identification of individual profiles will support promising therapeutic approaches based on target determination.

Lipopolysaccharide from Coxiella burnetii Is Involved in Bacterial Phagocytosis, Filamentous Actin Reorganization, and Inflammatory Responses through Toll-Like Receptor 4

The role of Toll-like receptors (TLRs) in the recognition of extracellular and facultative intracellular bacteria by the innate immune system has been extensively studied, but their role in the recognition of obligate intracellular organisms remains unknown. Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that specifically inhabits monocytes/macrophages. We showed in this study that C. burnetii LPS is involved in the uptake of virulent organisms by macrophages but not in that of avirulent variants. The uptake of virulent organisms was dependent on TLR4 because it was reduced in macrophages from TLR4−/− mice. In addition, LPS was responsible for filamentous actin reorganization induced by virulent C. burnetii, which was prevented in TLR4−/− macrophages. In contrast, the intracellular fate of C. burnetii was not affected in TLR4−/− macrophages, suggesting that TLR4 does not control the maturation of C. burnetii phagosome and the microbicidal activity of macrophages. These results are consistent with in vivo experiments because the pattern of tissue infection and the clearance of C. burnetii were similar in wild-type and TLR4−/− mice. We also showed that the number of granulomas was decreased in the liver of infected TLR4−/− mice, and the formation of splenic granulomas was only transient. The impaired formation of granulomas was associated with decreased production of IFN-γ and TNF. Taken together, these results demonstrate that TLR4 controls early events of C. burnetii infection such as macrophage phagocytosis, granuloma formation, and cytokine production.

Dysregulation of Cytokines in Acute Q Fever: Role of Interleukin-10 and Tumor Necrosis Factor in Chronic Evolution of Q Fever

Q fever manifests as primary infection or acute Q fever and may become chronic in patients with underlying valvulopathy. Because Coxiella burnetii infection depends on host response, we measured tumor necrosis factor (TNF), interleukin (IL)-6, IL-12, and IL-10 in patients with different clinical presentations of acute Q fever. Compared with control subjects, patients with uncomplicated acute Q fever exhibited increased release of the 4 cytokines. Their amounts were higher in patients with hepatitis than in patients with fever or pneumonia. In patients with valvulopathy, who exhibited the highest risk of chronic evolution, the amounts of TNF and IL-10 were higher than in patients without valvulopathy. TNF production was specifically enhanced in patients who developed Q fever endocarditis. These results show that acute Q fever is associated with cytokine overproduction. Persistent TNF amounts were associated with the occurrence of endocarditis in patients with valvulopathy, and that may be a marker of chronic evolution of Q fever.

Survival of Tropheryma whipplei, the Agent of Whipple's Disease, Requires Phagosome Acidification

ABSTRACT Tropheryma whipplei was established as the agent of Whipples disease in 2000, but the mechanisms by which it survives within host cells are still unknown. We show here that T. whipplei survives within HeLa cells by controlling the biogenesis of its phagosome. Indeed, T. whipplei colocalized with lysosome-associated membrane protein 1, a membrane marker of late endosomal and lysosomal compartments, but not with cathepsin D, a lysosomal hydrolase. This defect in phagosome maturation is specific to live organisms, since heat-killed bacilli colocalized with cathepsin D. In addition, T. whipplei survived within HeLa cells by adapting to acidic pH. The vacuoles containing T. whipplei were acidic (pH 4.7 ± 0.3) and acquired vacuolar ATPase, responsible for the acidic pH of late phagosomes. The treatment of HeLa cells with pH-neutralizing reagents, such as ammonium chloride, N-ethylmaleimide, bafilomycin A1, and chloroquine, increased the intravacuolar pH and promoted the killing of T. whipplei. The ability of T. whipplei to survive in an acidic environment and to interfere with phagosome-lysosome fusion is likely critical for its prolonged persistence in host cells during the course of Whipples disease. Our results suggest that manipulating the intravacuolar pH may provide a new approach for the treatment of Whipples disease.

Interleukin-10 Stimulates Coxiella burnetii Replication in Human Monocytes through Tumor Necrosis Factor Down-Modulation: Role in Microbicidal Defect of Q Fever

ABSTRACT Coxiella burnetii, an obligate intracellular bacterium, is the agent of Q fever. The chronic form of the disease is associated with the overproduction of interleukin-10 and deficient C. burnetii killing by monocytes. We hypothesized that the replication of C. burnetii inside monocytes requires a macrophage-deactivating cytokine such as interleukin-10. In the absence of interleukin-10, C. burnetii survived but did not replicate in monocytes. C. burnetii replication (measured 15 days) was induced in interleukin-10-treated monocytes. This effect of interleukin-10 is specific since transforming growth factor β1 had no effect on bacterial replication. C. burnetii replication involves the down-modulation of tumor necrosis factor (TNF) release. First, interleukin-10 suppressedC. burnetii-stimulated production of TNF. Second, the addition of recombinant TNF to interleukin-10-treated monocytes inhibited bacterial replication. Third, the incubation of infected monocytes with neutralizing anti-TNF antibodies favored C. burnetii replication. On the other hand, deficient C. burnetii killing by monocytes from patients with chronic Q fever involves interleukin-10. Indeed, C. burnetii replication was observed in monocytes from patients with Q fever endocarditis, but not in those from patients with acute Q fever. Bacterial replication was inhibited by neutralizing anti-interleukin-10 antibodies. As monocytes from patients with endocarditis overproduced interleukin-10, the defective bacterial killing is likely related to endogenous interleukin-10. These results suggest that interleukin-10 enables monocytes to support C. burnetii replication and to favor the development of chronic Q fever.

Impact of highly active anti-retroviral therapy (HAART) on cytokine production and monocyte subsets in HIV-infected patients

HIV infection is associated with cytokine production by monocytes and expansion of a monocyte subset that expresses high levels of CD16. Our study was designed to investigate the effects of anti‐retroviral therapies on these immune parameters. Four groups of HIV+ patients were included in the study. The first group comprised drug‐naive patients (n = 20); the second included patients who received two inhibitors of HIV reverse transcriptase (n = 45); the third group received a therapy combining these two inhibitors and one inhibitor of HIV protease (HAART) (n = 35); the fourth consisted of patients who had stopped their treatment (n = 20). The release of inflammatory cytokines (tumour necrosis factor, IL‐1β, IL‐6) and immunoregulatory cytokines such as IL‐10 by monocytes was determined by ELISA. The monocyte subsets expressing low or high levels of CD16 were studied by flow cytometry. Monocytes from patients naive of treatment released higher amounts of inflammatory cytokines and IL‐10 than HIV− individuals. Each anti‐retroviral therapy restored a normal pattern of cytokine secretion. Nevertheless, the release of cytokines increased again after the arrest of the treatment. The expansion of the monocyte subset that expresses high levels of CD16 was significantly decreased by HAART but not by the treatment including two inhibitors of reverse transcriptase. These results suggest that only HAART controls monocyte activation in the treatment of HIV infection.