TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS.

Cytoplasmic accumulation of TDP-43 is a disease hallmark for many cases of amyotrophic lateral sclerosis (ALS), associated with a neuroinflammatory cytokine profile related to upregulation of nuclear factor κB (NF-κB) and type I interferon (IFN) pathways. Here we show that this inflammation is driven by the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) when TDP-43 invades mitochondria and releases DNA via the permeability transition pore. Pharmacologic inhibition or genetic deletion of cGAS and its downstream signaling partner STING prevents upregulation of NF-κB and type I IFN induced by TDP-43 in induced pluripotent stem cell (iPSC)-derived motor neurons and in TDP-43 mutant mice. Finally, we document elevated levels of the specific cGAS signaling metabolite cGAMP in spinal cord samples from patients, which may be a biomarker of mtDNA release and cGAS/STING activation in ALS. Our results identify mtDNA release and cGAS/STING activation as critical determinants of TDP-43-associated pathology and demonstrate the potential for targeting this pathway in ALS.

Posttranslational Modification as a Critical Determinant of Cytoplasmic Innate Immune Recognition.

Cell surface innate immune receptors can directly detect a variety of extracellular pathogens to which cytoplasmic innate immune sensors are rarely exposed. Instead, within the cytoplasm, the environment is rife with cellular machinery and signaling pathways that are indirectly perturbed by pathogenic microbes to activate intracellular sensors, such as pyrin, NLRP1, NLRP3, or NLRC4. Therefore, subtle changes in key intracellular processes such as phosphorylation, ubiquitination, and other pathways leading to posttranslational protein modification are key determinants of innate immune recognition in the cytoplasm. This concept is critical to establish the "guard hypothesis" whereby otherwise homeostatic pathways that keep innate immune sensors at bay are released in response to alterations in their posttranslational modification status. Originally identified in plants, evidence that a similar guardlike mechanism exists in humans has recently been identified, whereby a mutation that prevents phosphorylation of the innate immune sensor pyrin triggers a dominantly inherited autoinflammatory disease. It is also noteworthy that even when a cytoplasmic innate immune sensor has a direct ligand, such as bacterial peptidoglycan (NOD1 or NOD2), RNA (RIG-I or MDA5), or DNA (cGAS or IFI16), it can still be influenced by posttranslational modification to dramatically alter its response. Therefore, due to their existence in the cytoplasmic milieu, posttranslational modification is a key determinant of intracellular innate immune receptor functionality.

Pyrin variant E148Q potentiates inflammasome activation and the effect of pathogenic mutations in cis.

OBJECTIVE: The p.E148Q variant in pyrin is present in different populations at a frequency of up to 29%, and has been associated with diseases, including vasculitis and FMF. The pathogenicity of p.E148Q in FMF is unclear, even when observed in cis or in trans to a single, typically recessive, pathogenic mutation. We performed functional validation to determine whether p.E148Q increases the ability of pyrin to form an active inflammasome complex in cell lines. METHODS: We interrogated the Australian Autoinflammatory Disease RegistrY (AADRY) to find candidate inheritance patterns for the p.E148Q variant in pyrin. Different pyrin variant combinations were tested in HEK293T cells stably expressing the adaptor protein apoptosis-associated speck-like (ASC), which were analysed by flow cytometry to visualize inflammasome formation, with and without stimulation by Clostridioides difficile toxin B (TcdB). Inflammasome-dependent cytokine secretion was also quantified by ELISA of supernatants from THP-1 cells transduced with lentiviral expression vectors. RESULTS: In AADRY, we observed the p.E148Q allele in individuals with autoinflammatory diseases alone or in conjunction with other pyrin variants. Two FMF families harboured the allele p.E148Q-M694I in cis with dominant heritability. In vitro, p.E148Q pyrin could spontaneously potentiate inflammasome formation, with increased IL-1ß and IL-18 secretion. p.E148Q in cis to classical FMF mutations provided significant potentiation of inflammasome formation. CONCLUSION: The p.E148Q variant in pyrin potentiates inflammasome activation in vitro. In cis, this effect is additive to known pathogenic FMF mutations. In some families, this increased effect could explain why FMF segregates as an apparently dominant disease.

Recessive NLRC4-Autoinflammatory Disease Reveals an Ulcerative Colitis Locus.

PURPOSE: NLRC4-associated autoinflammatory disease (NLRC4-AID) is an autosomal dominant condition presenting with a range of clinical manifestations which can include macrophage activation syndrome (MAS) and severe enterocolitis. We now report the first homozygous mutation in NLRC4 (c.478G > A, p.A160T) causing autoinflammatory disease with immune dysregulation and find that heterozygous carriers in the general population are at increased risk of developing ulcerative colitis. METHODS: Circulating immune cells and inflammatory markers were profiled and historical clinical data interrogated. DNA was extracted and sequenced using standard procedures. Inflammasome activation assays for ASC speck formation, pyroptosis, and IL-1ß/IL-18 secretion confirmed pathogenicity of the mutation in vitro. Genome-wide association of NLRC4 (A160T) with ulcerative colitis was examined using data from the IBD exomes portal. RESULTS: A 60-year-old Brazilian female patient was evaluated for recurrent episodes of systemic inflammation from six months of age. Episodes were characterized by recurrent low-grade fever, chills, oral ulceration, uveitis, arthralgia, and abdominal pain, followed by diarrhea with mucus and variable skin rash. High doses of corticosteroids were somewhat effective in controlling disease and anti-IL-1ß therapy partially controlled symptoms. While on treatment, serum IL-1ß and IL-18 levels remained elevated. Genetic investigations identified a homozygous mutation in NLRC4 (A160T), inherited in a recessive fashion. Increased ASC speck formation and IL-1ß/IL-18 secretion confirmed pathogenicity when NLRC4 (A160T) was analyzed in human cell lines. This allele is significantly enriched in patients with ulcerative colitis: OR 2.546 (95% 1.778-3.644), P = 0.01305. CONCLUSION: NLRC4 (A160T) can either cause recessively inherited autoinflammation and immune dysregulation, or function as a heterozygous risk factor for the development of ulcerative colitis.

Outcomes following SARS-CoV-2 infection in patients with primary and secondary immunodeficiency in the UK.

In March 2020, the United Kingdom Primary Immunodeficiency Network (UKPIN) established a registry of cases to collate the outcomes of individuals with PID and SID following SARS-CoV-2 infection and treatment. A total of 310 cases of SARS-CoV-2 infection in individuals with PID or SID have now been reported in the UK. The overall mortality within the cohort was 17.7% (n = 55/310). Individuals with CVID demonstrated an infection fatality rate (IFR) of 18.3% (n = 17/93), individuals with PID receiving IgRT had an IFR of 16.3% (n = 26/159) and individuals with SID, an IFR of 27.2% (n = 25/92). Individuals with PID and SID had higher inpatient mortality and died at a younger age than the general population. Increasing age, low pre-SARS-CoV-2 infection lymphocyte count and the presence of common co-morbidities increased the risk of mortality in PID. Access to specific COVID-19 treatments in this cohort was limited: only 22.9% (n = 33/144) of patients admitted to the hospital received dexamethasone, remdesivir, an anti-SARS-CoV-2 antibody-based therapeutic (e.g. REGN-COV2 or convalescent plasma) or tocilizumab as a monotherapy or in combination. Dexamethasone, remdesivir, and anti-SARS-CoV-2 antibody-based therapeutics appeared efficacious in PID and SID. Compared to the general population, individuals with PID or SID are at high risk of mortality following SARS-CoV-2 infection. Increasing age, low baseline lymphocyte count, and the presence of co-morbidities are additional risk factors for poor outcome in this cohort.

Monogenic autoinflammatory disorders: beyond the periodic fever.

The past two decades have seen an exponential increase in the number of monogenic autoinflammatory disorders described, coinciding with improved genetic sequencing techniques. This group of disorders has evolved to be heterogeneous and certainly more complex than the original four 'periodic fever syndromes' caused by innate immune over-activation. This review aims to provide an update on the classic periodic fever syndromes as well as introducing the broadening spectrum of clinical features seen in more recently described conditions.

Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)-LRR oligomerization interface.

BACKGROUND: Monogenic autoinflammatory disorders are characterized by dysregulation of the innate immune system, for example by gain-of-function mutations in inflammasome-forming proteins, such as NOD-like receptor family CARD-containing 4 protein (NLRC4). OBJECTIVE: Here we investigate the mechanism by which a novel mutation in the leucine-rich repeat (LRR) domain of NLRC4 (c.G1965C, p.W655C) contributes to autoinflammatory disease. METHODS: We studied 2 unrelated patients with early-onset macrophage activation syndrome harboring the same de novo mutation in NLRC4. In vitro inflammasome complex formation was quantified by using flow cytometric analysis of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 techniques and lentiviral transduction were used to generate THP-1 cells with either wild-type or mutant NLRC4 cDNA. Cell death and release of IL-1ß/IL-18 were quantified by using flow cytometry and ELISA, respectively. RESULTS: The p.W655C NLRC4 mutation caused increased ASC speck formation, caspase-1-dependent cell death, and IL-1ß/IL-18 production. ASC contributed to p.W655C NLRC4-mediated cytokine release but not cell death. Mutation of p.W655 activated the NLRC4 inflammasome complex by engaging with 2 interfaces on the opposing LRR domain of the oligomer. One key set of residues (p.D1010, p.D1011, p.L1012, and p.I1015) participated in LRR-LRR oligomerization when triggered by mutant NLRC4 or type 3 secretion system effector (PrgI) stimulation of the NLRC4 inflammasome complex. CONCLUSION: This is the first report of a mutation in the LRR domain of NLRC4 causing autoinflammatory disease. c.G1965C/p.W655C NLRC4 increased inflammasome activation in vitro. Data generated from various NLRC4 mutations provides evidence that the LRR-LRR interface has an important and previously unrecognized role in oligomerization of the NLRC4 inflammasome complex.

The classification, genetic diagnosis and modelling of monogenic autoinflammatory disorders.

Monogenic autoinflammatory disorders are an increasingly heterogeneous group of conditions characterised by innate immune dysregulation. Improved genetic sequencing in recent years has led not only to the discovery of a plethora of conditions considered to be 'autoinflammatory', but also the broadening of the clinical and immunological phenotypic spectra seen in these disorders. This review outlines the classification strategies that have been employed for monogenic autoinflammatory disorders to date, including the primary innate immune pathway or the dominant cytokine implicated in disease pathogenesis, and highlights some of the advantages of these models. Furthermore, the use of the term 'autoinflammatory' is discussed in relation to disorders that cross the innate and adaptive immune divide. The utilisation of next-generation sequencing (NGS) in this population is examined, as are potential in vivo and in vitro methods of modelling to determine pathogenicity of novel genetic findings. Finally, areas where our understanding can be improved are highlighted, such as phenotypic variability and genotype-phenotype correlations, with the aim of identifying areas of future research.

A novel Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis mutation further defines 14-3-3 binding of pyrin and distinction to Familial Mediterranean Fever.

OBJECTIVE: Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND) is a recently described monogenic autoinflammatory disease. The causal p.S242R MEFV mutation disrupts a binding motif of the regulatory 14-3-3 proteins within pyrin. Here, we investigate a family with clinical features consistent with PAAND in whom the novel p.E244K MEFV mutation, located in the +2 site of the 14-3-3 binding motif in pyrin, has been found. METHODS: Multiplex cytokine analyses were performed on p.E244K patient and control serum. Peripheral blood mononuclear cells were stimulated ex vivo with lipopolysaccharide (LPS). In vitro, inflammasome complex formation was evaluated by flow cytometry of Apoptosis-associated Speck-like protein containing a Caspase recruitment domain (ASC) specks. Interleukin-1ß (IL-1ß) and IL-18 production was quantified by ELISA. The ability of the p.E244K pyrin mutation to interact with 14-3-3 was assessed by immunoprecipitation. RESULTS: PAAND p.E244K patient serum displayed a different cytokine profile compared with patients with Familial Mediterranean Fever (FMF). In overexpression models, p.E244K pyrin was associated with decreased 14-3-3 binding and increased ASC speck formation. THP-1 monocytes expressing PAAND pyrin mutations demonstrated spontaneous caspase-1-dependent IL-1ß and IL-18 secretion, as well as cell death, which were significantly greater than those of wild-type and the FMF-associated mutation p.M694V. CONCLUSION: In PAAND, disruption of the +2 position of a 14-3-3 binding motif in pyrin results in its constitutive activation, with spontaneous production of IL-1ß and IL-18, associated with inflammatory cell death. The altered serum cytokine profile may explain the different clinical features exhibited by PAAND patients compared with those with FMF.

Need for intensive care in patients admitted for asthma: Red flags from the social history.

BACKGROUND AND OBJECTIVE: Asthma deaths in Australia are associated with illicit substance abuse, mental health problems and social issues. However, a large proportion of these deaths occurs out of hospital and is difficult to avert by the time the individuals seek medical attention. We hypothesized that these characteristics may also increase the risk for a patient to require intensive care admission when they present to emergency departments. METHODS: We studied consecutive patients admitted to a tertiary metropolitan hospital with a primary diagnosis of asthma between January 2010 and January 2014. Clinical and demographical data were obtained from chart review. The patient's postcode was used as a surrogate for socioeconomic status. RESULTS: There were 482 asthma patients admitted during the study period, of which 39 required intensive care. Ten patients admitted to intensive care (26%) used illicit drugs compared with 29 (7%) of those admitted to the ward (adjusted odds ratio: 3.6, P = 0.012). For illicit users, nonadherence to preventer therapy was associated with an even higher risk of intensive care unit admission. Socioeconomic index was lower in the group requiring intensive care admission. The frequency of psychiatric diagnoses was similar in both groups. CONCLUSION: Among patients admitted to hospital for asthma, illicit substance abuse is a strong independent risk factor for intensive care requirement. Preventer therapy nonadherence further increases this risk. Lower socioeconomic status is also associated with increased risk. These historical features should be actively sought on admission and may serve as useful 'red flags' to prompt consideration of intensive monitoring.

Monogenic autoinflammatory diseases: Cytokinopathies.

Rapid advances in genetics are providing unprecedented insight into functions of the innate immune system with identification of the mutations that cause monogenic autoinflammatory disease. Cytokine antagonism is profoundly effective in a subset of these conditions, particularly those associated with increased interleukin-1 (IL-1) activity, the inflammasomopathies. These include syndromes where the production of IL-1 is increased by mutation of innate immune sensors such as NLRP3, upstream signalling molecules such as PSTPIP1 and receptors or downstream signalling molecules, such as IL-1Ra. Another example of this is interferon (IFN) and the interferonopathies, with mutations in the sensors STING and MDA5, the upstream signalling regulator AP1S3, and a downstream inhibitor of IFN signalling, ISG15. We propose that this can be extended to cytokines such as IL-36, with mutations in IL-36Ra, and IL-10, with mutations in IL-10RA and IL-10RB, however mutations in sensors or upstream signalling molecules are yet to be described in these instances. Additionally, autoinflammatory diseases can be caused by multiple cytokines, for example with the activation of NF-κB/Rel, for which we propose the term Relopathies. This nosology is limited in that some cytokine pathways may be degenerate in their generation or execution, however provides insight into likely autoinflammatory disease candidates and the cytokines with which newly identified mutations may be associated, and therefore targeted.

Low circulating B cells in immunocompromised individuals are linked to poorer antibody responses to vaccines and a predisposition to viral infections.

Background: B cells play an important role in protection against viral infections, not only through the production of antibodies but also through their ability to act as antigen-presenting cells and produce cytokines. Objectives: To assess whether there is a link between low circulating B-cell counts and a predisposition to viral infections in immunocompromised individuals, we performed a retrospective cohort analysis at 2 National Health Service Clinical Immunology sites in England. Methods: Eligible patients were adults who were either diagnosed with or under investigation for an immunodeficiency and had recorded circulating B-cell counts. Information on viral infections was collected by using the departmental, hospital, and laboratory electronic information systems. A generalized linear model was used to analyze the relationship between B-cell counts and relevant indices of viral infection while controlling for patient age, diagnosis group, and T-cell and natural killer cell counts. Results: A total of 376 eligible patients were identified, 134 of whom had B-cell counts that were below the laboratory-defined refence range (<0.11 ×109/L). Patients with low numbers of circulating B cells had lower pretreatment immunoglobulin levels and poorer antibody responses to vaccines (Streptococcus pneumonia, Clostridium tetani, and Haemophilus influenzae type B). An increased number of chronic or recurrent (P = .001), severe or unusual (P = .001), and PCR-confirmed viral infections (P = .04) were recorded in these patients versus in those with normal numbers of circulating B cells. Conclusion: Overall, there was a statistically significant association between low circulating B-cell counts and the incidence of clinically important viral infections in this patient cohort, even when controlling for relevant covariates. Clinicians caring for patients with immunodeficiency should be vigilant for these types of infections, particularly in patients with low peripheral B-cell counts. A prospective study will be required to confirm these findings.

COVID-19 vaccine allergy advice and guidance: The experience of a UK tertiary referral centre.

Objective: The objective was to review COVID-19 vaccine allergy advice and guidance requests received and assess the impact of advice outcome on vaccination outcome. Design: A retrospective analysis of requests for advice and guidance regarding COVID-19 vaccine allergy was completed using an electronic referral system from February 2021 to January 2022. Participants: A total of 1265 independent patient requests for advice were received from primary care. Full vaccination information was available on 1210 patients who were included in the analysis. Main outcome measures: We evaluated the specific outcome of request for advice (written advice versus allergy consultation), rate of vaccination, vaccination combinations, and tolerance of vaccination. Results: Of the 1210 patients included, 959 (79%) were female. Eight hundred and ninety-six (74%) requests were managed with written advice only and of these 675 (75%) patients went on to be vaccinated. Overall, 891 (74%) of the population were vaccinated with 2 or more doses.Two hundred and nineteen patient consultations were undertaken with 109 (50%) prior to the first vaccination. Forty-nine (45%) consultations prior to vaccination were undertaken due to a label of anaphylaxis to vaccination in the past. Vaccination was recommended for all patients, and 78 (72%) of these received a first dose. Eight of these patients (10%) had symptoms within 1 h of vaccine administration.One hundred and ten (50%) consultations were undertaken for adverse reactions post COVID-19 vaccination, with 84 (76%) concerning immediate symptoms. Thirty patients (27%) who had a consultation had had adrenaline administered post vaccination. One patient had biopsy confirmed Stevens Johnson Syndrome and was referred to Dermatology. All others due for further doses (107 patients) were recommended to have subsequent doses with 49 (45%) offered the same vaccine. Eighty-nine patients had a vaccine administered post adverse reaction and 79 (88%) tolerated the dose.Skin testing and challenge to polyethylene glycol were negative in the 8 patients tested. Conclusions: Over 1000 requests for advice and guidance were received during the review period, managed mainly with written advice. The overwhelming majority of requests for advice and consultations were for females, with equal distribution both pre- and post-COVID-19 vaccine administration. Vaccination was recommended in all but 1 patient (with biopsy confirmed Stevens Johnson Syndrome). Polyethylene glycol allergy was not confirmed in any patient, nor did any patient have confirmed anaphylaxis when the vaccine was administered under our supervision, suggesting that type 1 mediated hypersensitivity is uncommon even in this "high risk" population.

Protein kinase R is an innate immune sensor of proteotoxic stress via accumulation of cytoplasmic IL-24.

Proteasome dysfunction can lead to autoinflammatory disease associated with elevated type I interferon (IFN-αß) and NF-κB signaling; however, the innate immune pathway driving this is currently unknown. Here, we identified protein kinase R (PKR) as an innate immune sensor for proteotoxic stress. PKR activation was observed in cellular models of decreased proteasome function and in multiple cell types from patients with proteasome-associated autoinflammatory disease (PRAAS). Furthermore, genetic deletion or small-molecule inhibition of PKR in vitro ameliorated inflammation driven by proteasome deficiency. In vivo, proteasome inhibitor-induced inflammatory gene transcription was blunted in PKR-deficient mice compared with littermate controls. PKR also acted as a rheostat for proteotoxic stress by triggering phosphorylation of eIF2α, which can prevent the translation of new proteins to restore homeostasis. Although traditionally known as a sensor of RNA, under conditions of proteasome dysfunction, PKR sensed the cytoplasmic accumulation of a known interactor, interleukin-24 (IL-24). When misfolded IL-24 egress into the cytosol was blocked by inhibition of the endoplasmic reticulum-associated degradation pathway, PKR activation and subsequent inflammatory signaling were blunted. Cytokines such as IL-24 are normally secreted from cells; therefore, cytoplasmic accumulation of IL-24 represents an internal danger-associated molecular pattern. Thus, we have identified a mechanism by which proteotoxic stress is detected, causing inflammation observed in the disease PRAAS.

Blood group type A secretors are associated with a higher risk of COVID-19 cardiovascular disease complications.

The SARS-CoV-2 virus causes COVID-19, an infection capable of causing severe disease and death but which can also be asymptomatic or oligosymptomatic. We investigated whether ABO blood group or secretor status was associated with COVID-19 severity. We investigated secretor status because expression of ABO glycans on secreted proteins and non-erythroid cells are controlled by a fucosyltransferase (FUT2), and inactivating FUT2 mutations result in a non-secretor phenotype which protects against some viral infections. Data combined from healthcare records and our own laboratory tests (n = 275) of hospitalized SARS-CoV-2 polymerase chain reaction positive patients confirmed higher than expected numbers of blood group A individuals compared to O (RR = 1.24, CI 95% [1.05, 1.47], p = 0.0111). There was also a significant association between group A and COVID-19-related cardiovascular complications (RR = 2.56, CI 95% [1.43, 4.55], p = 0.0011) which is independent of gender. Molecular analysis revealed that group A non-secretors are significantly less likely to be hospitalized than secretors. Testing of convalescent plasma donors, among whom the majority displayed COVID-19 symptoms and only a small minority required hospitalization, group A non-secretors were slightly over-represented. Our findings showed that group A non-secretors are not resistant to infection by SARS-CoV-2, but are more likely to experience a less severe form of associated disease.

TBK1 and IKKε Act Redundantly to Mediate STING-Induced NF-κB Responses in Myeloid Cells.

Stimulator of Interferon Genes (STING) is a critical component of host innate immune defense but can contribute to chronic autoimmune or autoinflammatory disease. Once activated, the cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS)-STING pathway induces both type I interferon (IFN) expression and nuclear factor-κB (NF-κB)-mediated cytokine production. Currently, these two signaling arms are thought to be mediated by a single upstream kinase, TANK-binding kinase 1 (TBK1). Here, using genetic and pharmacological approaches, we show that TBK1 alone is dispensable for STING-induced NF-κB responses in human and mouse immune cells, as well as in vivo. We further demonstrate that TBK1 acts redundantly with IκB kinase ε (IKKε) to drive NF-κB upon STING activation. Interestingly, we show that activation of IFN regulatory factor 3 (IRF3) is highly dependent on TBK1 kinase activity, whereas NF-κB is significantly less sensitive to TBK1/IKKε kinase inhibition. Our work redefines signaling events downstream of cGAS-STING. Our findings further suggest that cGAS-STING will need to be targeted directly to effectively ameliorate the inflammation underpinning disorders associated with STING hyperactivity.

Connexin-Dependent Transfer of cGAMP to Phagocytes Modulates Antiviral Responses.

Activation of cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in antiviral responses to many DNA viruses. Sensing of cytosolic DNA by cGAS results in synthesis of the endogenous second messenger cGAMP that activates stimulator of interferon genes (STING) in infected cells. Critically, cGAMP can also propagate antiviral responses to uninfected cells through intercellular transfer, although the modalities of this transfer between epithelial and immune cells remain poorly defined. We demonstrate here that cGAMP-producing epithelial cells can transactivate STING in cocultured macrophages through direct cGAMP transfer. cGAMP transfer was reliant upon connexin expression by epithelial cells and pharmacological inhibition of connexins blunted STING-dependent transactivation of the macrophage compartment. Macrophage transactivation by cGAMP contributed to a positive-feedback loop amplifying antiviral responses, significantly protecting uninfected epithelial cells against viral infection. Collectively, our findings constitute the first direct evidence of a connexin-dependent cGAMP transfer to macrophages by epithelial cells, to amplify antiviral responses.IMPORTANCE Recent studies suggest that extracellular cGAMP can be taken up by macrophages to engage STING through several mechanisms. Our work demonstrates that connexin-dependent communication between epithelial cells and macrophages plays a significant role in the amplification of antiviral responses mediated by cGAMP and suggests that pharmacological strategies aimed at modulating connexins may have therapeutic applications to control antiviral responses in humans.

The Salmonella pathogenicity island-2 subverts human NLRP3 and NLRC4 inflammasome responses.

Inflammasomes are signaling hubs that activate inflammatory caspases to drive cytokine maturation and cell lysis. Inflammasome activation by Salmonella Typhimurium infection or Salmonella-derived molecules is extensively studied in murine myeloid cells. Salmonella-induced inflammasome signaling in human innate immune cells, is however, poorly characterized. Here, we show that Salmonella mutation to inactivate the Salmonella pathogenicity island-2 type III secretion system (SPI2 T3SS) potentiates S. Typhimurium-induced inflammasome responses from primary human macrophages, resulting in strong IL-1ß production and macrophage death. Inactivation of the SPI1 T3SS diminished human macrophage responses to WT and ΔSPI2 Salmonella. Salmonella ΔSPI2 elicited a mixed inflammasome response from human myeloid cells, in which NLR family CARD-domain containing protein 4 (NLRC4) and NLR family PYRIN-domain containing protein 3 (NLRP3) perform somewhat redundant functions in generating IL-1ß and inducing pyroptosis. Our data suggest that Salmonella employs the SPI2 T3SS to subvert SPI1-induced NLRP3 and NLRC4 inflammasome responses in human primary macrophages, in a species-specific immune evasion mechanism.