Too Much of a Good Thing: Extended Duration of Gut Microbiota Depletion Reverses Protection From Experimental Autoimmune Uveitis.

Purpose: Using the model of experimental autoimmune uveitis (EAU) induced by immunization with a retinal antigen, two studies have reported contradictory results on disease development following oral antibiotic treatment (ABX). We showed that long-term ABX did not affect EAU, but another study showed that short-term ABX was protective. We therefore studied the effects of ABX on EAU, gut microbiota, and host immune responses as a function of treatment duration. Methods: EAU-susceptible mice were treated orally with broad-spectrum antibiotics starting at least 10 weeks (long-term) or 1 week (short-term) before immunization until termination of the experiment. Gut microbiota were characterized by 16S amplicon sequencing, and host gut immune elements were studied phenotypically and functionally. Results: Long-term ABX had no effect, whereas short-term ABX delayed EAU, as previously reported by us and others, respectively. Microbial sequencing revealed progressive reduction of gut microbiota that showed some differences in the two ABX groups. Interestingly, duration of ABX was associated with a gradual disappearance of the CD4+ and CD4+CD8+ subset of gut intraepithelial lymphocytes (IELs). This IEL subset is microbiota dependent and is absent in germ-free mice. Relative abundance of Lactobacillus reuteri correlated with the frequencies of CD4+CD8+ IELs. IELs suppressed antigen-specific activation of autoreactive T cells in culture. Conclusions: Gut microbiota may play dual roles in uveitis development: They promote EAU development but also help maintain IEL populations that have regulatory function against autoreactive T cells. We propose that the progressive loss of this population during long-term ABX reverses the EAU-ameliorating effects of microbiota depletion.

Microbiota as Drivers and as Therapeutic Targets in Ocular and Tissue Specific Autoimmunity.

Autoimmune uveitis is a major cause of blindness in humans. Activation of retina-specific autoreactive T cells by commensal microbiota has been shown to trigger uveitis in mice. Although a culprit microbe and/or its immunogenic antigen remains to be identified, studies from inducible and spontaneous mouse models suggest the potential of microbiota-modulating therapies for treating ocular autoimmune disease. In this review, we summarize recent findings on the contribution of microbiota to T cell-driven, tissue-specific autoimmunity, with an emphasis on autoimmune uveitis, and analyze microbiota-altering interventions, including antibiotics, probiotics, and microbiota-derived metabolites (e.g., short-chain fatty acids), which have been shown to be effective in other autoimmune diseases. We also discuss the need to explore more translational animal models as well as to integrate various datasets (microbiomic, transcriptomic, proteomic, metabolomic, and other cellular measurements) to gain a better understanding of how microbiota can directly or indirectly modulate the immune system and contribute to the onset of disease. It is hoped that deeper understanding of these interactions may lead to more effective treatment interventions.

Trypanosoma cruzi Neurotrophic Factor Facilitates Cardiac Repair in a Mouse Model of Chronic Chagas Disease.

Most patients acutely infected with Trypanosoma cruzi undergo short-term structural and functional cardiac alterations that heal without sequelae. By contrast, in patients whose disease progresses to chronic infection, irreversible degenerative chronic Chagas cardiomyopathy (CCC) may develop. To account for the contrast between cardiac regeneration in high-parasitism acute infection and progressive cardiomyopathy in low-parasitism CCC, we hypothesized that T. cruzi expresses repair factors that directly facilitate cardiac regeneration. We investigated, as one such repair factor, the T. cruzi parasite-derived neurotrophic factor (PDNF), known to trigger survival of cardiac myocytes and fibroblasts and upregulate chemokine chemokine C-C motif ligand 2, which promotes migration of regenerative cardiac progenitor cells (CPCs). Using in vivo and in vitro models of Chagas disease, we tested whether T. cruzi PDNF promotes cardiac repair. Quantitative PCR and flow cytometry of heart tissue revealed that stem-cell antigen-1 (Sca-1+) CPCs expand in acute infection in parallel to parasitism. Recombinant PDNF induced survival and expansion of ex vivo CPCs, and intravenous administration of PDNF into naïve mice upregulated mRNA of cardiac stem-cell marker Sca-1. Furthermore, in CCC mice, a 3-week intravenous administration of PDNF protocol induced CPC expansion and reversed left ventricular T-cell accumulation and cardiac remodeling including fibrosis. Compared with CCC vehicle-treated mice, which developed severe atrioventricular block, PDNF-treated mice exhibited reduced frequency and severity of conduction abnormalities. Our findings are in support of the novel concept that T. cruzi uses PDNF to promote mutually beneficial cardiac repair in Chagas disease. This could indicate a possible path to prevention or treatment of CCC.

Parasite-derived neurotrophic factor/trans-sialidase of Trypanosoma cruzi links neurotrophic signaling to cardiac innate immune response.

The Chagas' disease parasite Trypanosoma cruzi elicits a potent inflammatory response in acutely infected hearts that keeps parasitism in check and triggers cardiac abnormalities. A most-studied mechanism underlying innate immunity in T. cruzi infection is Toll-like receptor (TLR) activation by lipids and other parasite molecules. However, yet-to-be-identified pathways should exist. Here, we show that T. cruzi strongly upregulates monocyte chemoattractant protein 1 (MCP-1)/CCL2 and fractalkine (FKN)/CX3CL1 in cellular and mouse models of heart infection. Mechanistically, upregulation of MCP-1 and FKN stems from the interaction of parasite-derived neurotrophic factor (PDNF)/trans-sialidase with neurotrophic receptors TrkA and TrkC, as assessed by pharmacological inhibition, neutralizing antibodies, and gene silencing studies. Administration of a single dose of intravenous PDNF to naive mice results in a dose-dependent increase in MCP-1 and FKN in the heart and liver with pulse-like kinetics that peak at 3 h postinjection. Intravenous PDNF also augments MCP-1 and FKN in TLR signaling-deficient MyD88-knockout mice, underscoring the MyD88-independent action of PDNF. Although single PDNF injections do not increase MCP-1 and FKN receptors, multiple PDNF injections at short intervals up the levels of receptor transcripts in the heart and liver, suggesting that sustained PDNF triggers cell recruitment at infection sites. Thus, given that MCP-1 and FKN are chemokines essential to the recruitment of immune cells to combat inflammation triggers and to enhance tissue repair, our findings uncover a new mechanism in innate immunity against T. cruzi infection mediated by Trk signaling akin to an endogenous inflammatory and fibrotic pathway resulting from cardiomyocyte-TrkA recognition by matricellular connective tissue growth factor (CTGF/CCN2).

Trypanosoma cruzi coaxes cardiac fibroblasts into preventing cardiomyocyte death by activating nerve growth factor receptor TrkA.

RATIONALE: Cardiomyocytes express neurotrophin receptor TrkA that promotes survival following nerve growth factor (NGF) ligation. Whether TrkA also resides in cardiac fibroblasts (CFs) and underlies cardioprotection is unknown. OBJECTIVE: To test whether CFs express TrkA that conveys paracrine signals to neighbor cardiomyocytes using, as probe, the Chagas disease parasite Trypanosoma cruzi, which expresses a TrkA-binding neurotrophin mimetic, named PDNF. T. cruzi targets the heart, causing chronic debilitating cardiomyopathy in ∼30% patients. METHODS AND RESULTS: Basal levels of TrkA and TrkC in primary CFs are comparable to those in cardiomyocytes. However, in the myocardium, TrkA expression is significantly lower in fibroblasts than myocytes, and vice versa for TrkC. Yet T. cruzi recognition of TrkA on fibroblasts, preferentially over cardiomyocytes, triggers a sharp and sustained increase in NGF, including in the heart of infected mice or of mice administered PDNF intravenously, as early as 3-h post-administration. Further, NGF-containing T. cruzi- or PDNF-induced fibroblast-conditioned medium averts cardiomyocyte damage by H(2)O(2), in agreement with the previously recognized cardioprotective role of NGF. CONCLUSIONS: TrkA residing in CFs induces an exuberant NGF production in response to T. cruzi infection, enabling, in a paracrine fashion, myocytes to resist oxidative stress, a leading Chagas cardiomyopathy trigger. Thus, PDNF-TrkA interaction on CFs may be a mechanism orchestrated by T. cruzi to protect its heart habitat, in concert with the long-term (decades) asymptomatic heart parasitism that characterizes Chagas disease. Moreover, as a potent booster of cardioprotective NGF in vivo, PDNF may offer a novel therapeutic opportunity against cardiomyopathies.

Trypanosoma cruzi highjacks TrkC to enter cardiomyocytes and cardiac fibroblasts while exploiting TrkA for cardioprotection against oxidative stress.

Chronic Chagas cardiomyopathy (CCC), caused by the obligate intracellular protozoan parasite Trypanosoma cruzi, is a major cause of morbidity and mortality in Latin America. CCC begins when T. cruzi enters cardiac cells for intracellular multiplication and differentiation, a process that starts with recognition of host-cell entry receptors. However, the nature of these surface molecules and corresponding parasite counter-receptor(s) is poorly understood. Here we show that antibodies against neurotrophin (NT) receptor TrkC, but not against family members TrkA and TrkB, prevent T. cruzi from invading primary cultures of cardiomyocytes and cardiac fibroblasts. Invasion is also selectively blocked by the TrkC ligand NT-3, and by antagonists of Trk autophosphorylation and downstream signalling. Therefore, these results indicate that T. cruzi gets inside cardiomyocytes and cardiac fibroblasts by activating TrkC preferentially over TrkA. Accordingly, short hairpin RNA interference of TrkC (shTrkC), but not TrkA, selectively prevents T. cruzi from entering cardiac cells. Additionally, T. cruzi parasite-derived neurotrophic factor (PDNF)/trans-sialidase, a TrkC-binding protein, but not family member gp85, blocks entry dose-dependently, underscoring the specificity of PDNF as TrkC counter-receptor in cardiac cell invasion. In contrast to invasion, competitive and shRNA inhibition studies demonstrate that T. cruzi-PDNF recognition of TrkA, but not TrkC on primary cardiomyocytes and the cardiomyocyte cell line H9c2 protects the cells against oxidative stress. Thus, this study shows that T. cruzi via PDNF favours neurotrophin receptor TrkC for cardiac cell entry and TrkA for cardiomyocyte protection against oxidative stress, and suggests a new therapeutic opportunity in PDNF and/or fragments thereof for CCC therapy as entry inhibitors and/or cardioprotection agonists.

Neurotrophin receptor TrkC is an entry receptor for Trypanosoma cruzi in neural, glial, and epithelial cells.

Trypanosoma cruzi, the agent of Chagas' disease, infects a variety of mammalian cells in a process that includes multiple cycles of intracellular division and differentiation starting with host receptor recognition by a parasite ligand(s). Earlier work in our laboratory showed that the neurotrophin-3 (NT-3) receptor TrkC is activated by T. cruzi surface trans-sialidase, also known as parasite-derived neurotrophic factor (PDNF). However, it has remained unclear whether TrkC is used by T. cruzi to enter host cells. Here, we show that a neuronal cell line (PC12-NNR5) relatively resistant to T. cruzi became highly susceptible to infection when overexpressing human TrkC but not human TrkB. Furthermore, trkC transfection conferred an ∼3.0-fold intracellular growth advantage. Sialylation-deficient Chinese hamster ovarian (CHO) epithelial cell lines Lec1 and Lec2 also became much more permissive to T. cruzi after transfection with the trkC gene. Additionally, NT-3 specifically blocked T. cruzi infection of the TrkC-NNR5 transfectants and of naturally permissive TrkC-bearing Schwann cells and astrocytes, as did recombinant PDNF. Two specific inhibitors of Trk autophosphorylation (K252a and AG879) and inhibitors of Trk-induced MAPK/Erk (U0126) and Akt kinase (LY294002) signaling, but not an inhibitor of insulin-like growth factor 1 receptor, abrogated TrkC-mediated cell invasion. Antibody to TrkC blocked T. cruzi infection of the TrkC-NNR5 transfectants and of cells that naturally express TrkC. The TrkC antibody also significantly and specifically reduced cutaneous infection in a mouse model of acute Chagas' disease. TrkC is ubiquitously expressed in the peripheral and central nervous systems, and in nonneural cells infected by T. cruzi, including cardiac and gastrointestinal muscle cells. Thus, TrkC is implicated as a functional PDNF receptor in cell entry, independently of sialic acid recognition, mediating broad T. cruzi infection both in vitro and in vivo.

Leptospira interrogans binds to human cell surface receptors including proteoglycans.

Leptospirosis is a global public health problem, primarily in the tropical developing world. The pathogenic mechanisms of the causative agents, several members of the genus Leptospira, have been underinvestigated. The exception to this trend has been the demonstration of the binding of pathogenic leptospires to the extracellular matrix (ECM) and its components. In this work, interactions of Leptospira interrogans bacteria with mammalian cells, rather than the ECM, were examined. The bacteria bound more efficiently to the cells than to the ECM, and a portion of this cell-binding activity was attributable to attachment to glycosaminoglycan (GAG) chains of proteoglycans (PGs). Chondroitin sulfate B PGs appeared to be the primary targets of L. interrogans attachment, while heparan sulfate PGs were much less important. Inhibition of GAG/PG-mediated attachment resulted in partial inhibition of bacterial attachment, suggesting that additional receptors for L. interrogans await identification. GAG binding may participate in the pathogenesis of leptospirosis within the host animal. In addition, because GAGs are expressed on the luminal aspects of epithelial cells in the proximal tubules of the kidneys, this activity may play a role in targeting the bacteria to this critical site. Because GAGs are shed in the urine, GAG binding may also be important for transmission to new hosts through the environment.