Neonatal microbiota-epithelial interactions that impact infection.

Despite modern therapeutic developments and prophylactic use of antibiotics during birth or in the first few months of life, enteric infections continue to be a major cause of neonatal mortality and morbidity globally. The neonatal period is characterized by initial intestinal colonization with microbiota and concurrent immune system development. It is also a sensitive window during which perturbations to the environment or host can significantly impact colonization by commensal microbes. Extensive research has demonstrated that these early life alterations to the microbiota can lead to enhanced susceptibility to enteric infections and increased systemic dissemination in newborns. Various contributing factors continue to pose challenges in prevention and control of neonatal enteric infections. These include alterations in the gut microbiota composition, impaired immune response, and effects of maternal factors. In addition, there remains limited understanding for how commensal microbes impact host-pathogen interactions in newborns. In this review, we discuss the recent recognition of initial microbiota-epithelial interactions that occur in neonates and can regulate susceptibility to intestinal infection. These studies suggest the development of neonatal prophylactic or therapeutic regimens that include boosting epithelial defense through microbiota-directed interventions.

Corrigendum: Curdlan Limits Mycobacterium tuberculosis Survival Through STAT-1 Regulated Nitric Oxide Production.

[This corrects the article DOI: 10.3389/fmicb.2019.01173.].

Intestinal microbiota disruption limits the isoniazid mediated clearance of Mycobacterium tuberculosis in mice.

Tuberculosis (TB) continues to remain a global threat due to the emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains and toxicity associated with TB drugs. Intestinal microbiota has been reported to affect the host response to immunotherapy and drugs. However, how it affects the potency of first-line TB drug isoniazid (INH) is largely unknown. Here, we examined the impact of gut microbial dysbiosis on INH efficiency to kill Mtb. In this study, we employed in vivo mouse model, pretreated with broad-spectrum antibiotics (Abx) cocktail to disrupt their intestinal microbial population prior to Mtb infection and subsequent INH therapy. We demonstrated that microbiota disruption results in the impairment of INH-mediated Mtb clearance, and aggravated TB-associated tissue pathology. Further, it suppressed the innate immunity and reduced CD4 T-cell response against Mtb. Interestingly, a distinct shift of gut microbial profile was noted with abundance of Enterococcus and reduction of Lactobacillus and Bifidobacterium population. Our results show that the intestinal microbiota is crucial determinant in efficacy of INH to kill Mtb and impacts the host immune response against infection. This work provides an intriguing insight into the potential links between host gut microbiota and potency of INH.

Induction of autophagy through CLEC4E in combination with TLR4: an innovative strategy to restrict the survival of Mycobacterium tuberculosis.

Host-directed therapies are gaining considerable impetus because of the emergence of drug-resistant strains of pathogens due to antibiotic therapy. Therefore, there is an urgent need to exploit alternative and novel strategies directed at host molecules to successfully restrict infections. The C-type lectin receptor CLEC4E and Toll-like receptor TLR4 expressed by host cells are among the first line of defense in encountering pathogens. Therefore, we exploited signaling of macrophages through CLEC4E in association with TLR4 agonists (C4.T4) to control the growth of Mycobacterium tuberculosis (Mtb). We observed significant improvement in host immunity and reduced bacterial load in the lungs of Mtb-infected mice and guinea pigs treated with C4.T4 agonists. Further, intracellular killing of Mtb was achieved with a 10-fold lower dose of isoniazid or rifampicin in conjunction with C4.T4 than the drugs alone. C4.T4 activated MYD88, PtdIns3K, STAT1 and RELA/NFKB, increased lysosome biogenesis, decreased Il10 and Il4 gene expression and enhanced macroautophagy/autophagy. Macrophages from autophagy-deficient (atg5 knockout or Becn1 knockdown) mice showed elevated survival of Mtb. The present findings also unveiled the novel role of CLEC4E in inducing autophagy through MYD88, which is required for control of Mtb growth. This study suggests a unique immunotherapeutic approach involving CLEC4E in conjunction with TLR4 to restrict the survival of Mtb through autophagy. ABBREVIATIONS: 3MA: 3 methyladenine; AO: acridine orange; Atg5: autophagy related 5; AVOs: acidic vesicular organelles; BECN1: beclin 1, autophagy related; BMDMs: bone marrow derived macrophages; bw: body weight; C4.T4: agonists of CLEC4E (C4/TDB) and TLR4 (T4/ultra-pure-LPS); CFU: colony forming unit; CLEC4E/Mincle: C-type lectin domain family 4, member e; CLR: c-type lectin receptor; INH: isoniazid; LAMP1: lysosomal-associated membrane protein 1; MφC4.T4: Mtb-infected C4.T4 stimulated macrophages; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MDC: monodansylcadaverine; MTOR: mechanistic target of rapamycin kinase; MYD88: myeloid differentiation primary response 88; NFKB: nuclear factor of kappa light polypeptide gene enhance in B cells; NLR: NOD (nucleotide-binding oligomerization domain)-like receptors; PFA: paraformaldehyde; PPD: purified protein derivative; PtdIns3K: class III phosphatidylinositol 3-kinase; RELA: v-rel reticuloendotheliosis viral oncogene homolog A (avian); RIF: rifampicin; RLR: retinoic acid-inducible gene-I-like receptors; TDB: trehalose-6,6´-dibehenate; TLR4: toll-like receptor 4; Ultra-pure-LPS: ultra-pure lipopolysaccharide-EK; V-ATPase: vacuolar-type H+ ATPase.

Potential Role of Gut Microbiota in Induction and Regulation of Innate Immune Memory.

The gut microbiota significantly regulates the development and function of the innate and adaptive immune system. The attribute of immunological memory has long been linked only with adaptive immunity. Recent evidence indicates that memory is also present in the innate immune cells such as monocytes/macrophages and natural killer cells. These cells exhibit pattern recognition receptors (PRRs) that recognize microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs) expressed by the microbes. Interaction between PRRs and MAMPs is quite crucial since it triggers the sequence of signaling events and epigenetic rewiring that not only play a cardinal role in modulating the activation and function of the innate cells but also impart a sense of memory response. We discuss here how gut microbiota can influence the generation of innate memory and functional reprogramming of bone marrow progenitors that helps in protection against infections. This article will broaden our current perspective of association between the gut microbiome and innate memory. In the future, this knowledge may pave avenues for development and designing of novel immunotherapies and vaccination strategies.

Gut Microbiota Regulates Mincle Mediated Activation of Lung Dendritic Cells to Protect Against Mycobacterium tuberculosis.

Gut microbial components serve as ligand for various pattern recognition receptors (PRRs) present on immune cells and thereby regulates host immunity. Dendritic cells (DCs) are highly specialized innate cells involved in immune response to Mycobacterium tuberculosis (Mtb) infection. The gut-lung axis is a potential therapeutic target in tuberculosis; however, understanding of the innate immune mechanism underlying the interaction of gut microbiota and lung still remains obscure. We investigated if antibiotics (Abx) induced gut dysbiosis is able to affect the activation of innate receptor, macrophage inducible C-type lectin (mincle) in lungs during Mtb infection. We found that dysbiosis reduced the lung mincle expression with a concomitant increase in Mtb survival. Further, Abx diminished the effector and memory T cell population, while elevating frequency of regulatory T cells (Tregs) in the lungs. Here, we show that dysbiotic mice exhibited low mincle expression on lung DCs. These DCs with impaired phenotype and functions had reduced ability to activate naïve CD4 T cells, and thus unable to restrict Mtb survival. In vivo administration of trehalose-6,6-dibehenate (TDB: mincle ligand) efficiently rescued this immune defect by enhancing lung DCs function and subsequent T cell response. Further, gut microbial profiling revealed augmentation of Lactobacillus upon mincle stimulation in microbiota depleted animals. Accordingly, supplementation with Lactobacillus restored mincle expression on lung DCs along with anti-Mtb response. Our data demonstrate that gut microbiota is crucial to maintain DC-dependent lung immune response against Mtb, mediated by mincle. Abx interrupt this process to induce impaired T cell-response and increased susceptibility to Mtb.

Curdlan Limits Mycobacterium tuberculosis Survival Through STAT-1 Regulated Nitric Oxide Production.

Host-directed therapies have emerged as an innovative and promising approach in tuberculosis (TB) treatment due to the observed limitations of current TB regimen such as lengthy duration and emergence of drug resistance. Thus, we explored the role of curdlan (beta glucan polysaccharide) as a novel strategy to activate macrophages against Mycobacterium tuberculosis (Mtb). The aim of the study was to investigate the role of curdlan in restricting the Mtb growth both in vitro and in vivo. Further, the immunomodulatory potential of curdlan against Mtb and the underlying mechanism is largely unknown. We found that curdlan treatment enhanced the antigen presentation, pro-inflammatory cytokines, Mtb uptake and killing activity of macrophages. In vivo studies showed that curdlan therapy significantly reduced the Mtb burden in lung and spleen of mice. Administration of curdlan triggered the protective Th1 and Th17 immunity while boosting the central and effector memory response in Mtb infected mice. Curdlan mediated anti-Mtb activity is through signal transducer and activator of transcription-1 (STAT-1), which regulates nitric oxide (NO) production through inducible NO synthase (iNOS) induction; along with this activation of nuclear factor kappa B (NF-κB) was also evident in Mtb infected macrophages. Thus, we demonstrate that curdlan exerts effective anti-tuberculous activity anti-tuberculous activity. It can be used as a potential host-directed therapy against Mtb.

TLR-3 Stimulation Skews M2 Macrophages to M1 Through IFN-αß Signaling and Restricts Tumor Progression.

During tumor progression, macrophages shift their protective M1-phenotype to pro-tumorigenic M2-subtype. Therefore, conversion of M2 to M1 phenotype may be a potential therapeutic intervention. TLRs are important pathogen recognition receptors expressed by cells of the immune system. Recently, a crucial role of TLR-3 has been suggested in cancer. Consequently, in the current study, we defined the role of TLR-3 in the reversion of M2-macrophages to M1. We analyzed the role of TLR-3 stimulation for skewing M2-macrophages to M1 at mRNA and protein level through qRT-PCR, flow cytometry, western blotting, and ELISA. The effectiveness of TLR-3L stimulation to revert M2-macrophages to M1 was evaluated in the murine tumor model. To determine the role of IFN-αß signaling in vitro and in vivo, we used Ifnar1-/- macrophages and anti-IFN-αß antibodies, respectively. We observed upregulation of M1-specific markers MHC-II and costimulatory molecules like CD86, CD80, and CD40 on M2-macrophages upon TLR-3 stimulation. In contrast, reduced expression of M2-indicators CD206, Tim-3, and pro-inflammatory cytokines was noticed. The administration of TLR-3L in the murine tumor reverted the M2-macrophages to M1-phenotype and regressed the tumor growth. The mechanism deciphered for macrophage reversion and controlling the tumor growth is dependent on IFN-αß signaling pathway. The results indicate that the signaling through TLR-3 is important in protection against tumors by skewing M2-macrophages to protective M1-subtype.

Reinforcing the Functionality of Mononuclear Phagocyte System to Control Tuberculosis.

The mononuclear phagocyte system (MPS) constitutes dendritic cells, monocytes, and macrophages. This system contributes to various functions that are essential for maintaining homeostasis, activation of innate immunity, and bridging it with the adaptive immunity. Consequently, MPS is highly important in bolstering immunity against the pathogens. However, MPS is the frontline cells in destroying Mycobacterium tuberculosis (Mtb), yet the bacterium prefers to reside in the hostile environment of macrophages. Therefore, it may be very interesting to study the struggle between Mtb and MPS to understand the outcome of the disease. In an event when MPS predominates Mtb, the host remains protected. By contrast, the situation becomes devastating when the pathogen tames and tunes the host MPS, which ultimately culminates into tuberculosis (TB). Hence, it becomes extremely crucial to reinvigorate MPS functionality to overwhelm Mtb and eliminate it. In this article, we discuss the strategies to bolster the function of MPS by exploiting the molecules associated with the innate immunity and highlight the mechanisms involved to overcome the Mtb-induced suppression of host immunity. In future, such approaches may provide an insight to develop immunotherapeutics to treat TB.

Morbid Sequences Suggest Molecular Mimicry between Microbial Peptides and Self-Antigens: A Possibility of Inciting Autoimmunity.

Understanding etiology of autoimmune diseases has been a great challenge for designing drugs and vaccines. The pathophysiology of many autoimmune diseases may be attributed to molecular mimicry provoked by microbes. Molecular mimicry hypothesizes that a sequence homology between foreign and self-peptides leads to cross-activation of autoreactive T cells. Different microbial proteins are implicated in various autoimmune diseases, including multiple sclerosis, human type 1 diabetes, primary biliary cirrhosis and rheumatoid arthritis. It may be imperative to identify the microbial epitopes that initiate the activation of autoreactive T cells. Consequently, in the present study, we employed immunoinformatics tools to delineate homologous antigenic regions between microbes and human proteins at not only the sequence level but at the structural level too. Interestingly, many cross-reactive MHC class II binding epitopes were detected from an array of microbes. Further, these peptides possess a potential to skew immune response toward Th1-like patterns. The present study divulges many microbial target proteins, their putative MHC-binding epitopes, and predicted structures to establish the fact that both sequence and structure are two important aspects for understanding the relationship between molecular mimicry and autoimmune diseases. Such findings may enable us in designing potential immunotherapies to tolerize autoreactive T cells.

Bolstering Immunity through Pattern Recognition Receptors: A Unique Approach to Control Tuberculosis.

The global control of tuberculosis (TB) presents a continuous health challenge to mankind. Despite having effective drugs, TB still has a devastating impact on human health. Contributing reasons include the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), the AIDS-pandemic, and the absence of effective vaccines against the disease. Indeed, alternative and effective methods of TB treatment and control are urgently needed. One such approach may be to more effectively engage the immune system; particularly the frontline pattern recognition receptor (PRR) systems of the host, which sense pathogen-associated molecular patterns (PAMPs) of Mtb. It is well known that 95% of individuals infected with Mtb in latent form remain healthy throughout their life. Therefore, we propose that clues can be found to control the remainder by successfully manipulating the innate immune mechanisms, particularly of nasal and mucosal cavities. This article highlights the importance of signaling through PRRs in restricting Mtb entry and subsequently preventing its infection. Furthermore, we discuss whether this unique therapy employing PRRs in combination with drugs can help in reducing the dose and duration of current TB regimen.

Infergen Stimulated Macrophages Restrict Mycobacterium tuberculosis Growth by Autophagy and Release of Nitric Oxide.

IFN alfacon-1 (Infergen) is a synthetic form of Interferon (IFN)-α2b. Infergen has immunomodulatory activity and is effective against hepatitis C virus. However, the effect of Infergen (IFG) on Mycobacterium tuberculosis (Mtb) has not yet been reported. Therefore, for the first time, we have studied the influence of IFG in constraining the survival of Mtb in human macrophages. We observed that IFG significantly enhanced the maturation and activation of macrophages. Further, it substantially augmented the secretion of IL-6, nitric oxide (NO) and antigen uptake. Moreover, macrophages exhibited remarkably higher bactericidal activity, as evidenced by reduction in the Mtb growth. Infergen-mediated mechanism was different from the type-1 interferons; since it worked through the activation of NF-κB, phosphorylation of STAT-3 and Akt-PI3K that improved the bactericidal activity through autophagy and NO release. In future, IFG immunotherapy can be a novel strategy for treating patients and controlling TB.

Alteration in the Gut Microbiota Provokes Susceptibility to Tuberculosis.

The microbiota that resides in the gastrointestinal tract provides essential health benefits to the host. In particular, they regulate immune homeostasis. Recently, several evidences indicate that alteration in the gut microbial community can cause infectious and non-infectious diseases. Tuberculosis (TB) is the most devastating disease, inflicting mortality and morbidity. It remains unexplored, whether changes in the gut microbiota can provoke or prevent TB. In the current study, we have demonstrated the antibiotics driven changes in the gut microbial composition and their impact on the survival of Mycobacterium tuberculosis (Mtb) in the lungs, liver, and spleen of infected mice, compared to those with intact microbiota. Interestingly, dysbiosis of microbes showed significant increase in the bacterial burden in lungs and dissemination of Mtb to spleen and liver. Furthermore, elevation in the number of Tregs and decline in the pool of IFN-γ- and TNF-α-releasing CD4 T cells was noticed. Interestingly, fecal transplantation in the gut microbiota disrupted animals exhibited improved Th1 immunity and lesser Tregs population. Importantly, these animals displayed reduced severity to Mtb infection. This study for the first time demonstrated the novel role of gut microbes in the susceptibility to TB and its prevention by microbial implants. In future, microbial therapies may help in treating patients suffering from TB.

Triggering through NOD-2 Differentiates Bone Marrow Precursors to Dendritic Cells with Potent Bactericidal activity.

Dendritic cells (DCs) play a crucial role in bridging innate and adaptive immunity by activating naïve T cells. The role of pattern recognition receptors like Toll-Like Receptors and Nod-Like Receptors expressed on DCs is well-defined in the recognition of the pathogens. However, nothing is precisely studied regarding the impact of NOD-2 signaling during the differentiation of DCs. Consequently, we explored the role of NOD-2 signaling in the differentiation of DCs and therefore their capability to activate innate and adaptive immunity. Intriguingly, we observed that NOD-2 stimulated DCs (nDCs) acquired highly activated and matured phenotype and exhibited substantially greater bactericidal activity by robust production of nitric oxide. The mechanism involved in improving the functionality of nDCs was dependent on IFN-αß signaling, leading to the activation of STAT pathways. Furthermore, we also observed that STAT-1 and STAT-4 dependent maturation and activation of DCs was under the feedback mechanism of SOCS-1 and SOCS-3 proteins. nDCs acquired enhanced potential to activate chiefly Th1 and Th17 immunity. Taken together, these results suggest that nDCs can be exploited as an immunotherapeutic agent in bolstering host immunity and imparting protection against the pathogens.

Signaling through NOD-2 and TLR-4 Bolsters the T cell Priming Capability of Dendritic cells by Inducing Autophagy.

T cells play a cardinal role in mediating protection against intracellular pathogens like Mycobacterium tuberculosis (Mtb). It is important to understand the factors that govern the T cell response; thereby can modulate its activity. Dendritic cells (DCs) are the major player in initiation and augmentation of T cell response. Targeting DCs to induce their optimum maturation and activation can lead to a better T cell response. Interestingly, we observed that combinatorial signaling of DCs through NOD-2 and TLR-4 fortified better yield of IL-12p40/70, IL-6 and IFN-γ and upregulated the expression of CD40, CD80 and CD86 costimulatory molecules. Further, we noticed improved phagocytic capabilities of DCs. Furthermore, NOD-2 and TLR-4 induced autophagy in DCs, which enhanced the activation of T cells. This study signifies that NOD-2 and TLR-4 exhibit synergism in invigorating the activity of DCs. Consequently, this strategy may have significant immunotherapeutic potential in bolstering the function of DCs and thus improving the immunity against pathogens.