Infection-elicited microbiota promotes host adaptation to nutrient restriction.

The microbiota performs multiple functions vital to host fitness, including defense against pathogens and adaptation to dietary changes. Yet, how environmental challenges shape microbiota resilience to nutrient fluctuation remains largely unexplored. Here, we show that transient gut infection can optimize host metabolism toward the usage of carbohydrates. Following acute infection and clearance of the pathogen, mice gained more weight as a result of white adipose tissue expansion. Concomitantly, previously infected mice exhibited enhanced carbohydrate (glucose) disposal and insulin sensitivity. This metabolic remodeling depended on alterations to the gut microbiota, with infection-elicited Betaproteobacteria being sufficient to enhance host carbohydrate metabolism. Further, infection-induced metabolic alteration protected mice against stunting in the context of limited nutrient availability. Together, these results propose that alterations to the microbiota imposed by acute infection may enhance host fitness and survival in the face of nutrient restriction, a phenomenon that may be adaptive in settings where both infection burden and food precarity are prevalent.

Interleukin-27 (IL-27) Mediates Susceptibility to Visceral Leishmaniasis by Suppressing the IL-17-Neutrophil Response.

The relationship established between Leishmania infantum and the vertebrate host can lead to a self-healing infection or to the manifestation of visceral leishmaniasis, a chronic systemic infection associated with high rates of mortality. We hypothesized that regulatory cytokines, such as interleukin-27 (IL-27), play a role in susceptibility to L. infantum infection. IL-27 is a heterodimeric cytokine composed of IL-27p28 and EBi3 subunits which, when combined, bind to IL-27R, leading to STAT-1 and -3 activation, playing a role in the regulation of the immune response. We observed in this work that IL-27 regulates the Th1/Th17 profiles in a mouse model of visceral leishmaniasis (VL) caused by L. infantum We showed here that the pathogen recognition by endosomal Toll-like receptors triggers a type I interferon (IFN) response, which acts through the type I IFN receptor and interferon regulatory factor 1 to induce IL-27 production by macrophages. Furthermore, IL-27 plays a major regulatory role in vivo, because Ebi3(-/-) mice can efficiently control parasite replication despite reduced levels of IFN-γ compared to wild-type mice. On the other hand, the absence of Ebi3 leads to exacerbated IL-17A production in the infected organs as well as in a coculture system, suggesting a direct regulatory action of IL-27 during L. infantum infection. As a consequence of exacerbated IL-17A in Ebi3(-/-) mice, a greater neutrophil influx was observed in the target organs, playing a role in parasite control. Thus, this work unveiled the molecular steps of IL-27 production after L. infantum infection and demonstrated its regulatory role in the IL-17A-neutrophil axis.

Intravital imaging of three different microvascular beds in SARS-CoV-2-infected mice.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) enters the respiratory tract, where it infects the alveoli epithelial lining. However, patients have sequelae that extend well beyond the alveoli into the pulmonary vasculature and, perhaps, beyond to the brain and other organs. Because of the dynamic events within blood vessels, histology does not report platelet and neutrophil behavior. Because of the rapid nontranscriptional response of these cells, neither single-cell RNA sequencing nor proteomics report robustly on their critical behaviors. We used intravital microscopy in level-3 containment to examine the pathogenesis of SARS-CoV-2 within 3 organs in mice expressing human angiotensin converting enzyme 2 (ACE-2) ubiquitously (CAG-AC-70) or on epithelium (K18-promoter). Using a neon-green SARS-CoV-2, we observed both the epithelium and endothelium infected in AC70 mice but only the epithelium in K18 mice. There were increased neutrophils in the microcirculation but not in the alveoli of the lungs of AC70 mice. Platelets formed large aggregates in the pulmonary capillaries. Despite only neurons being infected within the brain, profound neutrophil adhesion forming the nidus of large platelet aggregates were observed in the cerebral microcirculation, with many nonperfused microvessels. Neutrophils breached the brain endothelial layer associated with a significant disruption of the blood-brain-barrier. Despite ubiquitous ACE-2 expression, CAG-AC-70 mice had very small increases in blood cytokine, no increase in thrombin, no infected circulating cells, and no liver involvement suggesting limited systemic effects. In summary, our imaging of SARS-CoV-2-infected mice gave direct evidence that there is a significant perturbation locally in the lung and brain microcirculation induced by local viral infection leading to increased local inflammation and thrombosis in these organs.

Neuron-associated macrophage proliferation in the sensory ganglia is associated with peripheral nerve injury-induced neuropathic pain involving CX3CR1 signaling.

Resident macrophages are distributed across all tissues and are highly heterogeneous due to adaptation to different tissue-specific environments. The resident macrophages of the sensory ganglia (sensory neuron-associated macrophages, sNAMs) are in close contact with the cell body of primary sensory neurons and might play physiological and pathophysiological roles. After peripheral nerve injury, there is an increase in the population of macrophages in the sensory ganglia, which have been implicated in different conditions, including neuropathic pain development. However, it is still under debate whether macrophage accumulation in the sensory ganglia after peripheral nerve injury is due to the local proliferation of resident macrophages or a result of blood monocyte infiltration. Here, we confirmed that the number of macrophages increased in the sensory ganglia after the spared nerve injury (SNI) model in mice. Using different approaches, we found that the increase in the number of macrophages in the sensory ganglia after SNI is a consequence of the proliferation of resident CX3CR1+ macrophages, which participate in the development of neuropathic pain, but not due to infiltration of peripheral blood monocytes. These proliferating macrophages are the source of pro-inflammatory cytokines such as TNF and IL-1b. In addition, we found that CX3CR1 signaling is involved in the sNAMs proliferation and neuropathic pain development after peripheral nerve injury. In summary, these results indicated that peripheral nerve injury leads to sNAMs proliferation in the sensory ganglia in a CX3CR1-dependent manner accounting for neuropathic pain development. In conclusion, sNAMs proliferation could be modulated to change pathophysiological conditions such as chronic neuropathic pain.

Meningeal dendritic cells drive neuropathic pain through elevation of the kynurenine metabolic pathway in mice.

Neuropathic pain is one of the most important clinical consequences of injury to the somatosensory system. Nevertheless, the critical pathophysiological mechanisms involved in neuropathic pain development are poorly understood. In this study, we found that neuropathic pain is abrogated when the kynurenine metabolic pathway (KYNPATH) initiated by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is ablated pharmacologically or genetically. Mechanistically, it was found that IDO1-expressing dendritic cells (DCs) accumulated in the dorsal root leptomeninges and led to an increase in kynurenine levels in the spinal cord. In the spinal cord, kynurenine was metabolized by kynurenine-3-monooxygenase-expressing astrocytes into the pronociceptive metabolite 3-hydroxykynurenine. Ultimately, 3-hydroxyanthranilate 3,4-dioxygenase-derived quinolinic acid formed in the final step of the canonical KYNPATH was also involved in neuropathic pain development through the activation of the glutamatergic N-methyl-D-aspartate receptor. In conclusion, these data revealed a role for DCs driving neuropathic pain development through elevation of the KYNPATH. This paradigm offers potential new targets for drug development against this type of chronic pain.

Microbiota and Microglia Interactions in ASD.

Autism spectrum disorders (ASD) are serious, highly variable neurodevelopmental disorders, commonly characterized by the manifestation of specific behavioral abnormalities, such as stereotypic behaviors and deficits in social skills, including communication. Although the neurobiological basis for ASD has attracted attention in recent decades, the role of microglial cells, which are the main resident myeloid cell population in the brain, is still controversial and underexplored. Microglia play several fundamental roles in orchestrating brain development and homeostasis. As such, alterations in the intrinsic functions of these cells could be one of the driving forces responsible for the development of various neurodevelopmental disorders, including ASD. Microglia are highly sensitive to environmental cues. Amongst the environmental factors known to influence their intrinsic functions, the gut microbiota has emerged as a central player, controlling both microglial maturation and activation. Strikingly, there is now compelling data suggesting that the intestinal microbiota can play a causative role in driving the behavioural changes associated with ASD. Not only is intestinal dysbiosis commonly reported in ASD patients, but therapies targeting the microbiome can markedly alleviate behavioral symptoms. Here we explore the emerging mechanisms by which altered microglial functions could contribute to several major etiological factors of ASD. We then demonstrate how pre- and postnatal environmental stimuli can modulate microglial cell phenotype and function, underpinning the notion that reciprocal interactions between microglia and intestinal microbes could play a crucial role in ASD aetiology.

Regulatory T cells counteract neuropathic pain through inhibition of the Th1 response at the site of peripheral nerve injury.

The inflammatory/immune response at the site of peripheral nerve injury participates in the pathophysiology of neuropathic pain. Nevertheless, little is known about the local regulatory mechanisms underlying peripheral nerve injury that counteracts the development of pain. Here, we investigated the contribution of regulatory T (Treg) cells to the development of neuropathic pain by using a partial sciatic nerve ligation model in mice. We showed that Treg cells infiltrate and proliferate in the site of peripheral nerve injury. Local Treg cells suppressed the development of neuropathic pain mainly through the inhibition of the CD4 Th1 response. Treg cells also indirectly reduced neuronal damage and neuroinflammation at the level of the sensory ganglia. Finally, we identified IL-10 signaling as an intrinsic mechanism by which Treg cells counteract neuropathic pain development. These results revealed Treg cells as important inhibitory modulators of the immune response at the site of peripheral nerve injury that restrains the development of neuropathic pain. In conclusion, the boosting of Treg cell function/activity might be explored as a possible interventional approach to reduce neuropathic pain development after peripheral nerve damage.

IL-27 Counteracts Neuropathic Pain Development Through Induction of IL-10.

Neuroimmune-glia interactions have been implicated in the development of neuropathic pain. Interleukin-27 (IL-27) is a cytokine that presents regulatory activity in inflammatory conditions of the central nervous system. Thus, we hypothesized that IL-27 would participate in the neuropathic pain process. Here, we found that neuropathic pain caused by peripheral nerve injury (spared nerve injury model; SNI), was enhanced in IL-27-deficient(-/-) mice, whereas nociceptive pain is similar to that of wild-type mice. SNI induced an increase in the expression of IL-27 and its receptor subunit (Wsx1) in the sensory ganglia and spinal cord. IL-27 receptor was expressed mainly in resident macrophage, microglia, and astrocytes of the sensory ganglia and spinal cord, respectively. Finally, we identify that the antinociceptive effect of IL-27 was not observed in IL-10-/- mice. These results provided evidence that IL-27 is a cytokine produced after peripheral nerve injury that counteracts neuropathic pain development through induction of the antinociceptive cytokine IL-10. In summary, our study unraveled the role of IL-27 as a regulatory cytokine that counteracts the development of neuropathic pain after peripheral nerve damage. In conclusion, they indicate that immunotherapies based on IL-27 could emerge as possible therapeutic approaches for the prevention of neuropathic pain development after peripheral nerve injury.

Corrigendum: Neospora Caninum Activates p38 MAPK as an Evasion Mechanism against Innate Immunity.

[This corrects the article DOI: 10.3389/fmicb.2016.01456.].

Frontline Science: Blood-circulating leukocytes fail to infiltrate the spinal cord parenchyma after spared nerve injury.

The development of neuropathic pain after peripheral nerve injury involves neuroimmune-glial interactions in the spinal cord. However, whether the development of neuropathic pain depends on the infiltration of peripheral immune cells, such as monocytes, into the spinal cord parenchyma after peripheral nerve damage remains unclear. Here, we used a combination of different techniques such as transgenic reporter mouse (Cx3cr1GFP/+ and Ccr2RFP/+ mice), bone marrow chimeric mice, and parabiosis to investigate this issue in spared nerve injury (SNI) model. Herein, we provided robust evidence that, although microglial cells are activated/proliferate at the dorsal horn of the spinal cord after SNI, peripheral hematopoietic cells (including monocytes) are not able to infiltrate into the spinal cord parenchyma. Furthermore, there was no evidence of CCR2 expression in intrinsic cells of the spinal cord. However, microglial cells activation/proliferation in the spinal cord and mechanical allodynia after SNI were reduced in Ccr2-deficient mice. These results suggest that blood-circulating leukocytes cells are not able to infiltrate the spinal cord parenchyma after distal peripheral nerve injury. Nevertheless, they indicate that CCR2-expressing cells might be indirectly regulating microglia activation/proliferation in the spinal cord after SNI. In conclusion, our study supports that CCR2 inhibition could be explored as an interventional approach to reduce microglia activation and consequently neuropathic pain development after peripheral nerve injury.

The NOD2 signaling in peripheral macrophages contributes to neuropathic pain development.

Neuropathic pain is one of the most important types of chronic pain. It is caused by neuronal damage. Clinical and experimental studies suggest a critical role for neuroimmune interactions in the development of neuropathic pain. In this article, we have shown that the cytoplasmic receptor Nod-like receptor-2, NOD2, and its adaptor-signaling molecule RIPK2 participate in the development of neuropathic pain after peripheral nerve injury (spared nerve injury model). The activation of NOD2 signaling in peripheral macrophage mediates the development of neuropathic pain through the production of pronociceptive cytokines (tumor necrosis factor and IL-1ß). This study found that peripheral nerve injury promoted a systemic increase in the NOD2 ligand. These results highlight a previously undetermined role for NOD2 signaling in the development of neuropathic pain, suggesting a new potential target for preventing neuropathic pain.

TGFß1 signaling sustains aryl hydrocarbon receptor (AHR) expression and restrains the pathogenic potential of TH17 cells by an AHR-independent mechanism.

The aryl hydrocarbon receptor (AHR) is a transcription factor activated by ligand highly expressed on TH17 cells, and AHR-deficient CD4+ T cells have impaired production of IL-17A and IL-22. Although AHR activation can exacerbate in vivo TH17 cell-mediated autoimmunity, accumulating data indicate that AHR is a nonpathogenic TH17 marker. Thus it remains unclear how AHR activation is regulated and impacts on the generation of TH17 subsets. Here we demonstrated that AHR pathway is activated during in vitro pathogenic TH17 polarization, but it is quickly downregulated. Under these conditions, additional AHR activation promoted IL-22 but not IL-17A. Interestingly, AHR high sustained expression and IL-17A promotion were only achieved when TGFß1 was present in the culture. In addition to the effect on AHR regulation, TGFß1 presented a dual role by simultaneously suppressing the TH17 pathogenic phenotype acquisition. This latter effect was independent of AHR stimulation, since its activation did not confer a TH17 anti-inflammatory profile and Ahr-/- cells did not upregulate any TH17 pathogenic marker. Through the use of EAE model, we demonstrated that AHR is still functional in encephalitogenic CD4+ T cells and the adoptive transfer of Ahr-/- TH17 cells to recipient mice resulted in milder EAE development when compared to their WT counterparts. Altogether, our data demonstrated that although AHR is highly expressed on in vitro-generated nonpathogenic TH17 cells, its ligation does not shift TH17 cells to an anti-inflammatory phenotype. Further studies investigating the role of AHR beyond TH17 differentiation may provide a useful understanding of the physiopathology of autoimmune diseases.

Canonical PI3Kγ signaling in myeloid cells restricts Trypanosoma cruzi infection and dampens chagasic myocarditis.

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi (T. cruzi) and is an important cause of severe inflammatory heart disease. However, the mechanisms driving Chagas disease cardiomyopathy have not been completely elucidated. Here, we show that the canonical PI3Kγ pathway is upregulated in both human chagasic hearts and hearts of acutely infected mice. PI3Kγ-deficient mice and mutant mice carrying catalytically inactive PI3Kγ are more susceptible to T. cruzi infection. The canonical PI3Kγ signaling in myeloid cells is essential to restrict T. cruzi heart parasitism and ultimately to avoid myocarditis, heart damage, and death of mice. Furthermore, high PIK3CG expression correlates with low parasitism in human Chagas' hearts. In conclusion, these results indicate an essential role of the canonical PI3Kγ signaling pathway in the control of T. cruzi infection, providing further insight into the molecular mechanisms involved in the pathophysiology of chagasic heart disease.

Nucleotide-binding oligomerization domain-containing protein 2 prompts potent inflammatory stimuli during Neospora caninum infection.

Neospora caninum is an apicomplexan parasite responsible for major economic losses due to abortions in cattle. Innate immune responses are crucial for host resistance against the infection, however the molecules involved in parasite recognition are still poorly understood. Nod2 is a cytosolic receptor that recognizes several pathogens and its role during N. caninum infection has not yet been described. In that sense, we evaluated the role of Nod2 in host response against this parasite. We found that infection of macrophages induced increased expression of Nod2, which colocalized with the parasites' vacuoles. Nod2-deficient macrophages showed an impaired induction of pro-inflammatory cytokines, increased production of modulatory molecules, and failure to restrict parasite replication. In vivo, Nod2-knockout mice showed a reduction of MAPK phosphorylation and proinflammatory cytokines, followed by decreased inflammation in target organs and increment in parasite burden. Surprisingly, these mice were partially resistant to lethal doses of tachyzoites. In addition, these phenomena were not observed in Rip2-/- mice. In conclusion, our study indicates that Nod2-dependent responses account for N. caninum elimination. On the other hand, the inflammatory milieu induced by this innate receptor provoked pathogenesis and death in severe experimental neosporosis.

Neospora caninum Activates p38 MAPK as an Evasion Mechanism against Innate Immunity.

Due to the high prevalence and economic impact of neosporosis, the development of safe and effective vaccines and therapies against this parasite has been a priority in the field and is crucial to limit horizontal and vertical transmission in natural hosts. Limited data is available regarding factors that regulate the immune response against this parasite and such knowledge is essential in order to understand Neospora caninum induced pathogenesis. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes, including growth, differentiation, apoptosis, and immune-mediated responses. In that sense, our goal was to understand the role of MAPKs during the infection by N. caninum. We found that p38 phosphorylation was quickly triggered in macrophages stimulated by live tachyzoites and antigen extracts, while its chemical inhibition resulted in upregulation of IL-12p40 production and augmented B7/MHC expression. In vivo blockade of p38 resulted in an amplified production of cytokines, which preceded a reduction in latent parasite burden and enhanced survival against the infection. Additionally, the experiments indicate that the p38 activation is induced by a mechanism that depends on GPCR, PI3K and AKT signaling pathways, and that the phenomena here observed is distinct that those induced by Toxoplasma gondii's GRA24 protein. Altogether, these results showed that N. caninum manipulates p38 phosphorylation in its favor, in order to downregulate the host's innate immune responses. Additionally, those results infer that active interference in this signaling pathway may be useful for the development of a new therapeutic strategy against neosporosis.