Tissue specific diversification, virulence and immune response to Mycobacterium bovis BCG in a patient with an IFN-γ R1 deficiency.

Summary: We characterized Mycobacterium bovis BCG isolates found in lung and brain samples from a previously vaccinated patient with IFNγR1 deficiency. The isolates collected displayed distinct genomic and phenotypic features consistent with host adaptation and associated changes in antibiotic susceptibility and virulence traits. Background: We report a case of a patient with partial recessive IFNγR1 deficiency who developed disseminated BCG infection after neonatal vaccination (BCG-vaccine). Distinct M. bovis BCG-vaccine derived clinical strains were recovered from the patient's lungs and brain. Methods: BCG strains were phenotypically (growth, antibiotic susceptibility, lipid) and genetically (whole genome sequencing) characterized. Mycobacteria cell infection models were used to assess apoptosis, necrosis, cytokine release, autophagy, and JAK-STAT signaling. Results: Clinical isolates BCG-brain and BCG-lung showed distinct Rv0667 rpoB mutations conferring high- and low-level rifampin resistance; the latter displayed clofazimine resistance through Rv0678 gene (MarR-like transcriptional regulator) mutations. BCG-brain and BCG-lung showed mutations in fadA2, fadE5, and mymA operon genes, respectively. Lipid profiles revealed reduced levels of PDIM in BCG-brain and BCG-lung and increased TAGs and Mycolic acid components in BCG-lung, compared to parent BCG-vaccine. In vitro infected cells showed that the BCG-lung induced a higher cytokine release, necrosis, and cell-associated bacterial load effect when compared to BCG-brain; conversely, both strains inhibited apoptosis and altered JAK-STAT signaling. Conclusions: During a chronic-disseminated BCG infection, BCG strains can evolve independently at different sites likely due to particular microenvironment features leading to differential antibiotic resistance, virulence traits resulting in dissimilar responses in different host tissues.

Immunologic characterization of 3 murine regimens of allergen-specific immunotherapy.

BACKGROUND: Allergen-specific immunotherapy (ASIT) is used to treat the symptoms of immediate type I hypersensitivity. The mechanisms driving establishment of allergen tolerance are not yet fully understood. OBJECTIVE: The goal of this study was to develop and immunologically characterize 3 murine models of ASIT to simulate protocols currently used to treat patients with type I hypersensitivities. METHODS: Ovalbumin (OVA)-sensitized mice were desensitized to OVA by means of repeated injections of OVA with a rapid, intermediate, or gradual protocol. After desensitization, mice were assessed for clinical sensitivity to OVA, and immunologic parameters were assessed. RESULTS: Mice in all treatment protocols displayed decreased vascular permeability in response to OVA challenge after desensitization. Circulating OVA-specific IgE levels, as well as basophil activation in response to OVA stimulation and IgE cross-linking, were significantly decreased in all treatment groups. Intermediate and gradual protocols, but not rapid desensitization, suppressed splenocyte proliferation and production of IL-4, IL-5, and IFN-γ in response to OVA and polyclonal activation. Similarly, significant increases in IL-10 production, numbers of CD4(+)CD25(+) forkhead box protein 3-positive regulatory T cells, and OVA-specific IgG1 antibody levels were only observed in mice undergoing prolonged ASIT regimens. CONCLUSION: Suppression of IgE-mediated activation is a common feature of all desensitization schedules. Induction of immunoregulatory networks requires prolonged desensitization schedules.

Basophils help establish protective immunity induced by irradiated larval vaccination for filariasis.

Basophils are increasingly recognized as playing important roles in the immune response toward helminths. In this study, we evaluated the role of basophils in vaccine-mediated protection against filariae, tissue-invasive parasitic nematodes responsible for diseases such as elephantiasis and river blindness. Protective immunity and immunological responses were assessed in BALB/c mice vaccinated with irradiated L3 stage larvae and depleted of basophils with weekly injections of anti-CD200R3 antibody. Depletion of basophils after administration of the vaccination regimen but before challenge infection did not alter protective immunity. In contrast, basophil depletion initiated prior to vaccination and continued after challenge infection significantly attenuated the protective effect conferred by vaccination. Vaccine-induced cellular immune responses to parasite antigen were substantially decreased in basophil-depleted mice, with significant decreases in CD4(+) T-cell production of IL-4, IL-5, IL-10, and IFN-γ. Interestingly, skin mast cell numbers, which increased significantly after vaccination with irradiated L3 larvae, were unchanged after vaccination in basophil-depleted mice. These findings demonstrate that basophils help establish the immune responses responsible for irradiated L3 vaccine protection.

Vaccination with intestinal tract antigens does not induce protective immunity in a permissive model of filariasis.

Antigens obtained from the intestinal tract of filarial nematodes have been proposed as potential safe and effective vaccine candidates. Because they may be 'hidden' from the immune response during natural infection, yet accessible by antibodies induced by vaccination, intestinal antigens may have a low potential for eliciting allergic responses when vaccinating previously infected individuals. Despite prior promising data, vaccination with intestinal antigens has yet to be tested in a permissive model of filariasis. In this study we investigated the efficacy of vaccination with filarial intestinal antigens in the permissive Litomosoides sigmodontis BALB/c model of filariasis, and we evaluated the extent to which these antigens are recognized by the immune system during and after infection. Infected BALB/c mice developed lower IgG antibody responses to soluble intestinal antigens (GutAg) than to soluble antigens of whole worms (LsAg). Similarly, GutAg induced less proliferation and less production of IL-4 and IFNγ from splenocytes of infected mice than LsAg. In contrast to these differences, active infection resulted in equivalent levels of circulating GutAg-specific IgE and LsAg-specific IgE levels. Consistent with this, basophil activation, as assessed by flow cytometric staining of intracellular basophil IL-4 expression, was equivalent in response to GutAg and LsAg. Vaccination with GutAg adsorbed to CpG/alum induced GutAg specific IgG1 and IgG2A production, with GutAg specific IgG titers greater than 5-fold higher than those measured in previously infected animals. Despite this response to GutAg vaccination, vaccinated mice harbored similar parasite burdens 8 weeks post infection when compared to non-vaccinated controls. These studies demonstrate that soluble antigens obtained from the intestinal tracts of L. sigmodontis have some qualities of 'hidden' antigens, but they still sensitize mice to allergic reactions and fail to protect against future infection when given as a vaccine adsorbed to alum/CPG.

Chronic helminth infection reduces basophil responsiveness in an IL-10-dependent manner.

Basophils play a key role in the development and effector phases of type 2 immune responses in both allergic diseases and helminth infections. This study shows that basophils become less responsive to IgE-mediated stimulation when mice are chronically infected with Litomosoides sigmodontis, a filarial nematode, and Schistosoma mansoni, a blood fluke. Although excretory/secretory products from microfilariae of L. sigmodontis suppressed basophils in vitro, transfer of microfilariae into mice did not result in basophil suppression. Rather, reduced basophil responsiveness, which required the presence of live helminths, was found to be dependent on host IL-10 and was accompanied by decreases in key IgE signaling molecules known to be downregulated by IL-10. Given the importance of basophils in the development of type 2 immune responses, these findings help explain the mechanism by which helminths protect against allergy and may have broad implications for understanding how helminth infections alter other disease states in people.

Chronic helminth infection does not exacerbate Mycobacterium tuberculosis infection.

BACKGROUND: Chronic helminth infections induce a Th2 immune shift and establish an immunoregulatory milieu. As both of these responses can suppress Th1 immunity, which is necessary for control of Mycobacterium tuberculosis (MTB) infection, we hypothesized that chronic helminth infections may exacerbate the course of MTB. METHODOLOGY/PRINCIPAL FINDINGS: Co-infection studies were conducted in cotton rats as they are the natural host for the filarial nematode Litomosoides sigmodontis and are an excellent model for human MTB. Immunogical responses, histological studies, and quantitative mycobacterial cultures were assessed two months after MTB challenge in cotton rats with and without chronic L. sigmodontis infection. Spleen cell proliferation and interferon gamma production in response to purified protein derivative were similar between co-infected and MTB-only infected animals. In contrast to our hypothesis, MTB loads and occurrence and size of lung granulomas were not increased in co-infected animals. CONCLUSIONS/SIGNIFICANCE: These findings suggest that chronic filaria infections do not exacerbate MTB infection in the cotton rat model. While these results suggest that filaria eradication programs may not facilitate MTB control, they indicate that it may be possible to develop worm-derived therapies for autoimmune diseases that do not substantially increase the risk for infections.

Helminth protection against autoimmune diabetes in nonobese diabetic mice is independent of a type 2 immune shift and requires TGF-ß.

Leading hypotheses to explain helminth-mediated protection against autoimmunity postulate that type 2 or regulatory immune responses induced by helminth infections in the host limit pathogenic Th1-driven autoimmune responses. We tested these hypotheses by investigating whether infection with the filarial nematode Litomosoides sigmodontis prevents diabetes onset in IL-4-deficient NOD mice and whether depletion or absence of regulatory T cells, IL-10, or TGF-ß alters helminth-mediated protection. In contrast to IL-4-competent NOD mice, IL-4-deficient NOD mice failed to develop a type 2 shift in either cytokine or Ab production during L. sigmodontis infection. Despite the absence of a type 2 immune shift, infection of IL-4-deficient NOD mice with L. sigmodontis prevented diabetes onset in all mice studied. Infections in immunocompetent and IL-4-deficient NOD mice were accompanied by increases in CD4(+)CD25(+)Foxp3(+) regulatory T cell frequencies and numbers, respectively, and helminth infection increased the proliferation of CD4(+)Foxp3(+) cells. However, depletion of CD25(+) cells in NOD mice or Foxp3(+) T cells from splenocytes transferred into NOD.scid mice did not decrease helminth-mediated protection against diabetes onset. Continuous depletion of the anti-inflammatory cytokine TGF-ß, but not blockade of IL-10 signaling, prevented the beneficial effect of helminth infection on diabetes. Changes in Th17 responses did not seem to play an important role in helminth-mediated protection against autoimmunity, because helminth infection was not associated with a decreased Th17 immune response. This study demonstrates that L. sigmodontis-mediated protection against diabetes in NOD mice is not dependent on the induction of a type 2 immune shift but does require TGF-ß.

Anti-FcεR1 antibody injections activate basophils and mast cells and delay Type 1 diabetes onset in NOD mice.

Mounting evidence suggests that helminth infections protect against autoimmune diseases. As helminths cause chronic IgE-mediated activation of basophils and mast cells we hypothesized that continuous activation of these cells could prevent diabetes onset in nonobese diabetic (NOD) mice in the absence of infection. Anti-FcεR1 activated basophils and mast cells and resulted in the release of IL-4 and histamine into the bloodstream. Anti-FcεR1-treated NOD mice showed a type 2 shift in insulin-specific antibody production and exhibited significant delays in diabetes onset. IL-4 responses played a partial role as the protective effect of anti-FcεR1 therapy was diminished in IL-4-deficient NOD mice. In contrast, histamine signaling was not required as anti-FcεR1-mediated protection was not reduced in mice treated with histamine receptor blockers. These results demonstrate that anti-FcεR1 therapy delays diabetes onset in NOD mice and suggest that chronic basophil and mast cell activation may represent a new avenue of therapy for Th1-associated autoimmune diseases.

Basophils amplify type 2 immune responses, but do not serve a protective role, during chronic infection of mice with the filarial nematode Litomosoides sigmodontis.

Chronic helminth infections induce a type 2 immune response characterized by eosinophilia, high levels of IgE, and increased T cell production of type 2 cytokines. Because basophils have been shown to be substantial contributors of IL-4 in helminth infections, and because basophils are capable of inducing Th2 differentiation of CD4(+) T cells and IgE isotype switching in B cells, we hypothesized that basophils function to amplify type 2 immune responses in chronic helminth infection. To test this, we evaluated basophil function using the Litomosoides sigmodontis filaria model of chronic helminth infection in BALB/c mice. Time-course studies showed that eosinophilia, parasite Ag-specific CD4(+) T cell production of IL-4 and IL-5 and basophil activation and IL-4 production in response to parasite Ag all peak late (6-8 wk) in the course of L. sigmodontis infection, after parasite-specific IgE has become detectable. Mixed-gender and single-sex worm implantation experiments demonstrated that the relatively late peak of these responses was not dependent on the appearance of circulating microfilariae, but may be due to initial low levels of parasite Ag load and/or habitation of the developing worms in the pleural space. Depletion of basophils throughout the course of L. sigmodontis infection caused significant decreases in total and parasite-specific IgE, eosinophilia, and parasite Ag-driven CD4(+) T cell proliferation and IL-4 production, but did not alter total worm numbers. These results demonstrate that basophils amplify type 2 immune responses, but do not serve a protective role, in chronic infection of mice with the filarial nematode L. sigmodontis.

Induction of type 2 responses by schistosome worms during prepatent infection.

During natural schistosome infection, the induction of T helper type 2 (Th2) responses has been ascribed to parasite eggs, because exposure of the host to this life-cycle stage elicits a polarized Th2 response to egg antigens. In the present study, we show that schistosome worms also elicit systemic, antigen-specific type 2 responses during prepatent infection, before egg deposition begins. CD4(+) T cells producing interleukin (IL)-4 were induced by both male and female worms during single-sex infections, demonstrating that this response is independent of exposure to eggs. The Th2 response was accompanied by production of immunoglobulin E and the sensitization of circulating basophils to produce additional IL-4 in response to schistosome antigens. Together, our data show that schistosome worms establish an immunologic milieu where CD4(+) T cells and basophils are both primed to produce IL-4 before eggs are laid, suggesting that worms play a role in establishment of the Th2 response that is critical for host survival and parasite transmission.

Type 2 immune-inducing helminth vaccination maintains protective efficacy in the setting of repeated parasite exposures.

Animal studies have demonstrated that helminth vaccines which induce type 2 immune responses can be protective. To date, however, such vaccines have not been tested against repeated parasite challenges. Since repeated antigenic challenge of patients with allergic disease results in immunologic tolerance, we hypothesized that a helminth vaccine which induces type 2 immune responses may lose its protective efficacy in the setting of repeated parasite exposures (RPEs). To test this hypothesis, we examined whether RPEs induce immunological tolerance and reduce the effectiveness of a type 2 immune-inducing vaccine. BALB/c mice vaccinated against Litomosoides sigmodontis, a filarial nematode of rodents, were repeatedly exposed to irradiated larvae for 2 or 8 weeks or to non-irradiated infectious larvae for three months. Vaccination-induced parasite-specific IgE levels, parasite antigen-driven basophil interleukin 4 (IL-4) release, and Th2 skewing of the cellular immune response remained stable in the face of RPEs. Furthermore, RPEs in vaccinated mice did not augment immunoregulatory responses, as parasite antigen-driven cellular proliferation, production of IL-10, and frequencies of CD4(+)CD25(+)FoxP3(+) regulatory T-cells were not altered by RPEs. Challenge infections with infectious L3-stage larvae resulted in lower worm burdens in vaccinated mice given RPEs than in vaccinated controls. These results demonstrate that vaccines which induce type 2 immune responses can maintain their efficacy in the setting of repeated parasite exposures.

Litomosoides sigmodontis: a simple method to infect mice with L3 larvae obtained from the pleural space of recently infected jirds (Meriones unguiculatus).

Litomosoides sigmodontis is a filarial nematode that is used as a mouse model for human filarial infections. The life cycle of L. sigmodontis comprises rodents as definitive hosts and tropical rat mites as alternate hosts. Here, we describe a method of infecting mice with third stage larvae (L3) extracted from the pleural space of recently infected jirds (Meriones unguiculatus). This method enables infection of mice with a known number of L3 larvae without the time-consuming dissection of L3 larvae from mites and results in higher worm recovery and patency rates than conventional methods. Additionally, this method allows for geographical separation of the facility maintaining the L. sigmodontis life cycle from the institution at which mice are infected.

Stat1 deficiency in the host enhances interleukin-12-mediated tumor regression.

Signal transducer and activator of transcription 1 (Stat1) is considered a key transcription factor that inhibits tumorigenesis, and Stat1 activation in the host is required for interleukin-12 (IL-12)-mediated generation of CTL activity. Using syngeneic Stat1-/- C3H mice bearing SCCVII tumors in this study, we discovered opposite results. Stat1 deficiency in the host significantly enhances IL-12-mediated tumor regression, resulting in tumor eradication from 60% of SCCVII tumor-bearing mice and significant inhibition of tumor growth when compared with control treatment (P < 0.01). This effect is independent of both Stat1-activating cytokine IFN-gamma and Stat1-downstream effector molecule FasL because neither neutralization of IFN-gamma nor knocking out of FasL enhances or inhibits IL-12-mediated tumor regression. IL-12 induces a high intensity of tumor-specific CTL activity in Stat1-deficient mice (P < 0.01), increases the CD8 T-cell density in tumor bearing Stat1-/- mice, and induces a T-cell-dependent tumor regression. The increased CTL activity and the high-intensity infiltration of T cells into the tumors in IL-12-treated Stat1-/- mice are likely due to the longer survival than the same cells from wild-type mice. Together, the data show that inhibition of Stat1 expression in the host enhances tumor-local IL-12 gene therapy for regressing tumors. This conclusion provides a new concept for designing an effective treatment strategy.

Regression of high-grade malignancy in mice by bleomycin and interleukin-12 electrochemogenetherapy.

PURPOSE: Bleomycin electrochemotherapy has been successfully used in preclinical studies and clinical trials for treating squamous cell carcinoma (SCC) and adenocarcinoma; however, it is not effective for treating recurrent tumors or metastatic tumors, or for preventing tumor redevelopment. In this study, we explore the coadministration of bleomycin and interleukin-12 (IL-12) followed by electroporation for treating primary and metastatic tumors. EXPERIMENTAL DESIGN: Bleomycin, IL-12 plasmid DNA, or a combination of both were injected into high-grade malignant mammary tumors and SCCVII followed by electroporation. The tumor growth, survival, metastasis in lungs, CTL activity, and vascular density were analyzed. The results were analyzed by the two-sided Student's t test and Gehan's Wilcoxon test. RESULTS: Coadministration of bleomycin and IL-12 via electroporation eradicates preestablished 4T1 mammary tumors in up to 60% of mice, inhibits metastatic tumor development, and extends the long-term survival. Likewise, coadministration of bleomycin and IL-12 via electroporation eradicates squamous cell carcinoma (SCCVII) in 100% of mice and prevents tumor redevelopment in 80% of mice. Neither bleomycin nor IL-12 alone is able to achieve the same therapeutic potency. The primary role of bleomycin is to inhibit the tumor vessel development; the primary role of IL-12 is to increase the immune response that extends the survival of treated mice and inhibits the tumor redevelopment. CONCLUSIONS: This combination modality has great potential to be translated in a clinical setting for treating high-grade malignancies and for preventing tumor redevelopment.