Regulation of neonatal IgA production by the maternal microbiota.

Infants are prone to enteric infections due to an underdeveloped immune system. The maternal microbiota, through shaping the neonatal microbiota, helps establish a strong immune system in infants. We and others have observed the phenomenon of enhanced early neonatal immunoglobulin A (IgA) production in preweaning immunocompetent mice nursed by immunodeficient dams. Here, we show that this enhancement of IgA in neonates results from maternally derived microbiota. In addition, we have found that the neonatal IgA production can be induced by Lactobacillus reuteri, which is enriched in the milk of immunodeficient dams. Moreover, we show that while the production of neonatal IgA is dependent on neonatal T cells, the immunodeficient maternal microbiota-mediated enhancement of neonatal IgA has a T cell-independent component. Indeed, this enhancement may be dependent on type 3 innate lymphoid cells in the neonatal small intestinal lamina propria. Interestingly, maternal microbiota-induced neonatal IgA does not cross-react with common enteric pathogens. Future investigations will determine the functional consequences of having this extra IgA.

Peripheral loss of EphA4 ameliorates TBI-induced neuroinflammation and tissue damage.

BACKGROUND: The continuum of pro- and anti-inflammatory response elicited by traumatic brain injury (TBI) is suggested to play a key role in the outcome of TBI; however, the underlying mechanisms remain ill -defined. METHODS: Here, we demonstrate that using bone marrow chimeric mice and systemic inhibition of EphA4 receptor shifts the pro-inflammatory milieu to pro-resolving following acute TBI. RESULTS: EphA4 expression is increased in the injured cortex as early as 2 h post-TBI and on CX3CR1gfp-positive cells in the peri-lesion. Systemic inhibition or genetic deletion of EphA4 significantly reduced cortical lesion volume and shifted the inflammatory profile of peripheral-derived immune cells to pro-resolving in the damaged cortex. These findings were consistent with in vitro studies showing EphA4 inhibition or deletion altered the inflammatory state of LPS-stimulated monocyte/macrophages towards anti-inflammatory. Phosphoarray analysis revealed that EphA4 may regulate pro-inflammatory gene expression by suppressing the mTOR, Akt, and NF-κB pathways. Our human metadata analysis further demonstrates increased EPHA4 and pro-inflammatory gene expression, which correlates with reduced AKT concurrent with increased brain injury severity in patients. CONCLUSIONS: Overall, these findings implicate EphA4 as a novel mediator of cortical tissue damage and neuroinflammation following TBI.

An orphaned Mce-associated membrane protein of Mycobacterium tuberculosis is a virulence factor that stabilizes Mce transporters.

Mycobacterium tuberculosis proteins that are exported out of the bacterial cytoplasm are ideally positioned to be virulence factors; however, the functions of individual exported proteins remain largely unknown. Previous studies identified Rv0199 as an exported membrane protein of unknown function. Here, we characterized the role of Rv0199 in M. tuberculosis virulence using an aerosol model of murine infection. Rv0199 appears to be a member of a Mce-associated membrane (Mam) protein family leading us to rename it OmamA, for orphaned Mam protein A. Consistent with a role in Mce transport, we showed OmamA is required for cholesterol import, which is a Mce4-dependent process. We further demonstrated a function for OmamA in stabilizing protein components of the Mce1 transporter complex. These results indicate a function of OmamA in multiple Mce transporters and one that may be analogous to the role of VirB8 in stabilizing Type IV secretion systems, as structural similarities between Mam proteins and VirB8 proteins are predicted by the Phyre 2 program. In this study, we provide functional information about OmamA and shed light on the function of Mam family proteins in Mce transporters.

Beyond the inflammasome: regulatory NOD-like receptor modulation of the host immune response following virus exposure.

A complex interaction exists between elements of the host innate immune system and viral pathogens. It is essential that the host mount a robust immune response during viral infection and effectively resolve inflammation once the pathogen has been eliminated. Members of the nucleotide-binding domain leucine-rich repeat [NBD-LRR; known as NOD-like receptor (NLR)] family of cytosolic pattern-recognition receptors are essential components of these immunological processes and have diverse functions in the host antiviral immune response. NLRs can be subgrouped based on their general function. The inflammasome-forming subgroup of NLRs are the best-characterized family members, and several have been found to modulate the maturation of IL-1ß and IL-18 following virus exposure. However, the members of the regulatory NLR subgroups are significantly less characterized. These NLRs uniquely function to modulate signalling pathways initiated by other families of pattern-recognition receptors, such as Toll-like receptors and/or Rig-I-like helicase receptors. Regulatory NLRs that augment pro-inflammatory pathways include nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and NOD2, which have been shown to form a multiprotein complex termed the NODosome that significantly modulates IFN and NF-κB signalling following viral infection. Conversely, a second subgroup of regulatory NLRs functions to negatively regulate inflammation. These inhibitory NLRs include NLRX1, NLRP12 and NLRC3, which have been shown to interact with TRAF molecules and various kinases to modulate diverse cellular processes. Targeting NLR signalling following infection with a virus represents a novel and promising therapeutic strategy. However, significant effort is still required to translate the current understanding of NLR biology into effective therapies.

Lactobacillus rhamnosus GG modulates innate signaling pathway and cytokine responses to rotavirus vaccine in intestinal mononuclear cells of gnotobiotic pigs transplanted with human gut microbiota.

BACKGROUND: A better understanding of mechanisms underlying dose-effects of probiotics in their applications as treatments of intestinal infectious or inflammatory diseases and as vaccine adjuvant is needed. In this study, we evaluated the modulatory effects of Lactobacillus rhamnosus GG (LGG) on transplanted human gut microbiota (HGM) and on small intestinal immune cell signaling pathways in gnotobiotic pigs vaccinated with an oral attenuated human rotavirus (AttHRV) vaccine. RESULTS: Neonatal HGM transplanted pigs were given two doses of AttHRV on 5 and 15 days of age and were divided into three groups: none-LGG (AttHRV), 9-doses LGG (AttHRV + LGG9X), and 14-doses LGG (AttHRV + LGG14X) (n = 3-4). At post-AttHRV-inoculation day 28, all pigs were euthanized and intestinal contents and ileal tissue and mononuclear cells (MNC) were collected. AttHRV + LGG14X pigs had significantly increased LGG titers in the large intestinal contents and shifted structure of the microbiota as indicated by the formation of a cluster that is separated from the cluster formed by the AttHRV and AttHRV + LGG9X pigs. The increase in LGG titers concurred with significantly increased ileal HRV-specific IFN-γ producing T cell responses to the AttHRV vaccine reported in our previous publication, suggesting pro-Th1 adjuvant effects of the LGG. Both 9- and 14-doses LGG fed pig groups had significantly higher IkBα level and p-p38/p38 ratio, while significantly lower p-ERK/ERK ratio than the AttHRV pigs, suggesting activation of regulatory signals during immune activation. However, 9-doses, but not 14-doses LGG fed pigs had enhanced IL-6, IL-10, TNF-α, TLR9 mRNA levels, and p38 MAPK and ERK expressions in ileal MNC. Increased TLR9 mRNA was in parallel with higher mRNA levels of cytokines, p-NF-kB and higher p-p38/p38 ratio in MNC of the AttHRV + LGG9X pigs. CONCLUSIONS: The relationship between modulation of gut microbiota and regulation of host immunity by different doses of probiotics is complex. LGG exerted divergent dose-dependent effects on the intestinal immune cell signaling pathway responses, with 9-doses LGG being more effective in activating the innate immunostimulating TLR9 signaling pathway than 14-doses in the HGM pigs vaccinated with AttHRV.

Nlrp12 deficiency alters gut microbiota and ameliorates Faslpr -mediated systemic autoimmunity in male mice.

NLRP12 has dual roles in shaping inflammation. We hypothesized that NLRP12 would modulate myeloid cells and T cell function to control systemic autoimmunity. Contrary to our hypothesis, the deficiency of Nlrp12 in autoimmune-prone B6.Faslpr/lpr mice ameliorated autoimmunity in males but not females. Nlrp12 deficiency dampened B cell terminal differentiation, germinal center reaction, and survival of autoreactive B cells leading to decreased production of autoantibodies and reduced renal deposition of IgG and complement C3. In parallel, Nlrp12 deficiency reduced the expansion of potentially pathogenic T cells, including double-negative T cells and T follicular helper cells. Furthermore, reduced pro-inflammatory innate immunity was observed, where the gene deletion decreased in-vivo expansion of splenic macrophages and mitigated ex-vivo responses of bone marrow-derived macrophages and dendritic cells to LPS stimulation. Interestingly, Nlrp12 deficiency altered the diversity and composition of fecal microbiota in both male and female B6/lpr mice. Notably, however, Nlrp12 deficiency significantly modulated small intestinal microbiota only in male mice, suggesting that the sex differences in disease phenotype might be gut microbiota-dependent. Together, these results suggest a potential pathogenic role of NLRP12 in promoting systemic autoimmunity in males. Future studies will investigate sex-based mechanisms through which NLRP12 differentially modulates autoimmune outcomes.

Deletion of ripA alleviates suppression of the inflammasome and MAPK by Francisella tularensis.

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1ß, IL-18, and TNF-α by resting macrophages. IL-1ß and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1ß and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis.

Exploration of Novel Pathways Underlying Irreversible Electroporation Induced Anti-Tumor Immunity in Pancreatic Cancer.

Advancements in medical sciences and technologies have significantly improved the survival of many cancers; however, pancreatic cancer remains a deadly diagnosis. This malignancy is often diagnosed late in the disease when metastases have already occurred. Additionally, the location of the pancreas near vital organs limits surgical candidacy, the tumor's immunosuppressive environment limits immunotherapy success, and it is highly resistant to radiation and chemotherapy. Hence, clinicians and patients alike need a treatment paradigm that reduces primary tumor burden, activates systemic anti-tumor immunity, and reverses the local immunosuppressive microenvironment to eventually clear distant metastases. Irreversible electroporation (IRE), a novel non-thermal tumor ablation technique, applies high-voltage ultra-short pulses to permeabilize targeted cell membranes and induce cell death. Progression with IRE technology and an array of research studies have shown that beyond tumor debulking, IRE can induce anti-tumor immune responses possibly through tumor neo-antigen release. However, the success of IRE treatment (i.e. full ablation and tumor recurrence) is variable. We believe that IRE treatment induces IFNγ expression, which then modulates immune checkpoint molecules and thus leads to tumor recurrence. This indicates a co-therapeutic use of IRE and immune checkpoint inhibitors as a promising treatment for pancreatic cancer patients. Here, we review the well-defined and speculated pathways involved in the immunostimulatory effects of IRE treatment for pancreatic cancer, as well as the regulatory pathways that may negate these anti-tumor responses. By defining these underlying mechanisms, future studies may identify improvements to systemic immune system engagement following local tumor ablation with IRE and beyond.

Type I Interferon Response Is Mediated by NLRX1-cGAS-STING Signaling in Brain Injury.

Background: Inflammation is a significant contributor to neuronal death and dysfunction following traumatic brain injury (TBI). Recent evidence suggests that interferons may be a key regulator of this response. Our studies evaluated the role of the Cyclic GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) signaling pathway in a murine model of TBI. Methods: Male, 8-week old wildtype, STING knockout (-/-), cGAS -/-, and NLRX1 -/- mice were subjected to controlled cortical impact (CCI) or sham injury. Histopathological evaluation of tissue damage was assessed using non-biased stereology, which was complemented by analysis at the mRNA and protein level using qPCR and western blot analysis, respectively. Results: We found that STING and Type I interferon-stimulated genes were upregulated after CCI injury in a bi-phasic manner and that loss of cGAS or STING conferred neuroprotection concomitant with a blunted inflammatory response at 24 h post-injury. cGAS -/- animals showed reduced motor deficits 4 days after injury (dpi), and amelioration of tissue damage was seen in both groups of mice up to 14 dpi. Given that cGAS requires a cytosolic damage- or pathogen-associated molecular pattern (DAMP/PAMP) to prompt downstream STING signaling, we further demonstrate that mitochondrial DNA is present in the cytosol after TBI as one possible trigger for this pathway. Recent reports suggest that the immune modulator NLR containing X1 (NLRX1) may sequester STING during viral infection. Our findings show that NLRX1 may be an additional regulator that functions upstream to regulate the cGAS-STING pathway in the brain. Conclusions: These findings suggest that the canonical cGAS-STING-mediated Type I interferon signaling axis is a critical component of neural tissue damage following TBI and that mtDNA may be a possible trigger in this response.

Corrigendum: Type I Interferon Response Is Mediated by NLRX1-cGAS-STING Signaling in Brain Injury.

[This corrects the article DOI: 10.3389/fnmol.2022.852243.].

Histotripsy Ablation in Preclinical Animal Models of Cancer and Spontaneous Tumors in Veterinary Patients: A Review.

New therapeutic strategies are direly needed in the fight against cancer. Over the last decade, several tumor ablation strategies have emerged as stand-alone or combination therapies. Histotripsy is the first completely noninvasive, nonthermal, and nonionizing tumor ablation method. Histotripsy can produce consistent and rapid ablations, even near critical structures. Additional benefits include real-time image guidance, high precision, and the ability to treat tumors of any predetermined size and shape. Unfortunately, the lack of clinically and physiologically relevant preclinical cancer models is often a significant limitation with all focal tumor ablation strategies. The majority of studies testing histotripsy for cancer treatment have focused on small animal models, which have been critical in moving this field forward and will continue to be essential for providing mechanistic insight. While these small animal models have notable translational value, there are significant limitations in terms of scale and anatomical relevance. To address these limitations, a diverse range of large animal models and spontaneous tumor studies in veterinary patients have emerged to complement existing rodent models. These models and veterinary patients are excellent at providing realistic avenues for developing and testing histotripsy devices and techniques designed for future use in human patients. Here, we provide a review of animal models used in preclinical histotripsy studies and compare histotripsy ablation in these models using a series of original case reports across a broad spectrum of preclinical animal models and spontaneous tumors in veterinary patients.

Immunological Effects of Histotripsy for Cancer Therapy.

Cancer is the second leading cause of death worldwide despite major advancements in diagnosis and therapy over the past century. One of the most debilitating aspects of cancer is the burden brought on by metastatic disease. Therefore, an ideal treatment protocol would address not only debulking larger primary tumors but also circulating tumor cells and distant metastases. To address this need, the use of immune modulating therapies has become a pillar in the oncology armamentarium. A therapeutic option that has recently emerged is the use of focal ablation therapies that can destroy a tumor through various physical or mechanical mechanisms and release a cellular lysate with the potential to stimulate an immune response. Histotripsy is a non-invasive, non-ionizing, non-thermal, ultrasound guided ablation technology that has shown promise over the past decade as a debulking therapy. As histotripsy therapies have developed, the full picture of the accompanying immune response has revealed a wide range of immunogenic mechanisms that include DAMP and anti-tumor mediator release, changes in local cellular immune populations, development of a systemic immune response, and therapeutic synergism with the inclusion of checkpoint inhibitor therapies. These studies also suggest that there is an immune effect from histotripsy therapies across multiple murine tumor types that may be reproducible. Overall, the effects of histotripsy on tumors show a positive effect on immunomodulation.

Histotripsy for the Treatment of Cholangiocarcinoma Liver Tumors: In Vivo Feasibility and Ex Vivo Dosimetry Study.

Histotripsy is a noninvasive, nonionizing, and nonthermal focused ultrasound ablation method that is currently being developed for the treatment of liver cancer. Promisingly, histotripsy has been shown for ablating primary [hepatocellular carcinoma (HCC)] and metastatic [colorectal liver metastasis (CLM)] liver tumors in preclinical and early clinical studies. The feasibility of treating cholangiocarcinoma (CC), a less common primary liver tumor that arises from the bile ducts, has not been explored previously. Given that prior work has established that histotripsy susceptibility is based on tissue mechanical properties, there is a need to explore histotripsy as a treatment for CC due to its dense fibrotic stromal components. In this work, we first investigated the feasibility of histotripsy for ablating CC tumors in vivo in a patient-derived xenograft mouse model. The results showed that histotripsy could generate CC tumor ablation using a 1-MHz small animal histotripsy system with treatment doses of 250, 500, and 1000 pulses/point. The second set of experiments compared the histotripsy doses required to ablate CC tumors to HCC and CLM tumors ex vivo. For this, human tumor samples were harvested after surgery and treated ex vivo with a 700-kHz clinical histotripsy transducer. Results demonstrated that significantly higher treatment doses were required to ablate CC and CLM tumors compared to HCC, with the highest treatment dose required for CC tumors. Overall, the results of this study suggest that histotripsy has the potential to be used for the ablation of CC tumors while also highlighting the need for tumor-specific treatment strategies.

Histotripsy Ablation Alters the Tumor Microenvironment and Promotes Immune System Activation in a Subcutaneous Model of Pancreatic Cancer.

Pancreatic cancer is a significant cause of cancer-related deaths in the United States with an abysmal five-year overall survival rate that is under 9%. Reasons for this mortality include the lack of late-stage treatment options and the immunosuppressive tumor microenvironment. Histotripsy is an ultrasound-guided, noninvasive, nonthermal tumor ablation therapy that mechanically lyses targeted cells. To study the effects of histotripsy on pancreatic cancer, we utilized an in vitro model of pancreatic adenocarcinoma and compared the release of potential antigens following histotripsy treatment to other ablation modalities. Histotripsy was found to release immune-stimulating molecules at magnitudes similar to other nonthermal ablation modalities and superior to thermal ablation modalities, which corresponded to increased innate immune system activation in vivo. In subsequent in vivo studies, murine Pan02 tumors were grown in mice and treated with histotripsy. Flow cytometry and rtPCR were used to determine changes in the tumor microenvironment over time compared to untreated animals. In mice with pancreatic tumors, we observed significantly increased tumor-progression-free and general survival, with increased activation of the innate immune system 24 h posttreatment and decreased tumor-associated immune cell populations within 14 days of treatment. This study demonstrates the feasibility of using histotripsy for pancreatic cancer ablation and provides mechanistic insight into the initial innate immune system activation following treatment. Further work is needed to establish the mechanisms behind the immunomodulation of the tumor microenvironment and immune effects.

The Pro-Inflammatory Chemokines CXCL9, CXCL10 and CXCL11 Are Upregulated Following SARS-CoV-2 Infection in an AKT-Dependent Manner.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a 'cytokine storm.' In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.

Antibiotics ameliorate lupus-like symptoms in mice.

Gut microbiota and the immune system interact to maintain tissue homeostasis, but whether this interaction is involved in the pathogenesis of systemic lupus erythematosus (SLE) is unclear. Here we report that oral antibiotics given during active disease removed harmful bacteria from the gut microbiota and attenuated SLE-like disease in lupus-prone mice. Using MRL/lpr mice, we showed that antibiotics given after disease onset ameliorated systemic autoimmunity and kidney histopathology. They decreased IL-17-producing cells and increased the level of circulating IL-10. In addition, antibiotics removed Lachnospiraceae and increased the relative abundance of Lactobacillus spp., two groups of bacteria previously shown to be associated with deteriorated or improved symptoms in MRL/lpr mice, respectively. Moreover, we showed that the attenuated disease phenotype could be recapitulated with a single antibiotic vancomycin, which reshaped the gut microbiota and changed microbial functional pathways in a time-dependent manner. Furthermore, vancomycin treatment increased the barrier function of the intestinal epithelium, thus preventing the translocation of lipopolysaccharide, a cell wall component of Gram-negative Proteobacteria and known inducer of lupus in mice, into the circulation. These results suggest that mixed antibiotics or a single antibiotic vancomycin ameliorate SLE-like disease in MRL/lpr mice by changing the composition of gut microbiota.

Non-Inflammasome Forming NLRs in Inflammation and Tumorigenesis.

Aberrant inflammation is an enabling characteristic of tumorigenesis. Thus, signaling cascades that alter inflammatory activation and resolution are of specific relevance to disease pathogenesis. Pattern recognition receptors (PRRs) are essential mediators of the host immune response and have emerged as critical elements affecting multiple facets of tumor pathobiology. The nucleotide-binding domain and leucine-rich repeat containing (NLR) proteins are intracellular PRRs that sense microbial and non-microbial products. Members of the NLR family can be divided into functional sub-groups based on their ability to either positively or negatively regulate the host immune response. Recent studies have identified a novel sub-group of non-inflammasome forming NLRs that negatively regulate diverse biological pathways associated with both inflammation and tumorigenesis. Understanding the mechanisms underlying the function of these unique NLRs will assist in the rationale design of future therapeutic strategies targeting a wide spectrum of inflammatory diseases and cancer. Here, we will discuss recent findings associated with this novel NLR sub-group and mechanisms by which these PRRs may function to alter cancer pathogenesis.