The immunogenic Heat shock proteins (HSPs) gp96, hsp70 and calreticulin bind to CD91 on antigen presenting cells for cross-presentation of the HSP-chaperoned peptides. This event leads to priming of T cell responses. We show that CD91 serves as a signaling receptor for these HSPs allowing for the maturation of the antigen presenting cells (APC), secretion of cytokines, and priming of T helper cells. Specifically, CD91 is phosphorylated in response to HSPs in a unique pattern and phospho-CD91 triggers signaling cascades to activate NF-κB. Each HSP-CD91 interaction on antigen presenting cells stimulates a unique cytokine profile which dictates priming of specific T helper cell subsets. Thus, in a TGF-β tumor microenvironment, immunization with calreticulin but not gp96 or hsp70 primes Th17 cell responses in a CD91-dependent manner. These results are important for development of T cell responses in situ in tumor-bearing hosts and for vaccination against cancer and infectious disease.

Mycobacterium tuberculosis truncated hemoglobin, HbN, is endowed with a potent nitric-oxide dioxygenase activity and has been found to relieve nitrosative stress and enhance in vivo survival of a heterologous host, Salmonella enterica Typhimurium, within the macrophages. These findings implicate involvement of HbN in the defense of M. tuberculosis against nitrosative stress. The protein carries a tunnel system composed of a short and a long tunnel branch that has been proposed to facilitate diatomic ligand migration to the heme and an unusual Pre-A motif at the N terminus, which does not contribute significantly to the structural integrity of the protein, as it protrudes out of the compact globin fold. Strikingly, deletion of Pre-A region from the M. tuberculosis HbN drastically reduces its ability to scavenge nitric oxide (NO), whereas its insertion at the N terminus of Pre-A lacking HbN of Mycobacterium smegmatis improved its nitric-oxide dioxygenase activity. Titration of the oxygenated adduct of HbN and its mutants with NO indicated that the stoichiometric oxidation of protein is severalfold slower when the Pre-A region is deleted in HbN. Molecular dynamics simulations show that the excision of Pre-A motif results in distinct changes in the protein dynamics, which cause the gate of the tunnel long branch to be trapped into a closed conformation, thus impeding migration of diatomic ligands toward the heme active site. The present study, thus, unequivocally demonstrates vital function of Pre-A region in NO scavenging and unravels its unique role by which HbN might attain its efficient NO-detoxification ability.Unlike many pathogens that are overtly harmful to their host, Mycobacterium tuberculosis can persist for years within humans in a clinically latent state. The success of tubercle bacillus to establish long term persistent infection within the human host lies in its ability to survive and resist hazardous environment of its intracellular niche. Early infection events involve entry and multiplication within the bacteriostatic environment of macrophages (1, 2), where an inducible nitric-oxide synthase generates copious amounts of nitric oxide (NO), which plays an important role in the host defense against microbial pathogens (3, 4) and restricts their growth and survival by inhibiting key enzymes such as the terminal respiratory oxidases (5) and ironsulfur centers of key enzymes such as aconitase (6, 7). In addition, NO combines with superoxide produced by respiring cells to form the highly oxidizing agent peroxynitrite (8). An efficient NO scavenging system, therefore, is required by microbial pathogens to cope with NO poisoning during their intracellular regime.M. tuberculosis has evolved resistance mechanisms by which toxic effects of NO and nitrosative stress can be evaded. One of the unique defense mechanisms by which it can protect itself from reactive nitrogen species relies on the oxygenated form of a group I truncated hemoglobin, HbN, which very efficiently converts NO into harmless nitrate (9 -11)...

Studies over the past decade have revealed a central role for innate immune sensors in autoimmune and autoinflammatory diseases. cGAS, a cytosolic DNA sensor, detects both foreign and host DNA and generates a second-messenger cGAMP, which in turn binds and activates stimulator of IFN genes (STING), leading to induction of type I interferons and inflammatory cytokines. Recently, gain-offunction mutations in STING have been identified in patients with STING-associated vasculopathy with onset in infancy (SAVI). SAVI patients present with early-onset systemic inflammation and interstitial lung disease, resulting in pulmonary fibrosis and respiratory failure. Here, we describe two independent SAVI mouse models, harboring the two most common mutations found in patients. A direct comparison of these strains reveals a hierarchy of immune abnormalities, lung inflammation and fibrosis, which do not depend on either IFN-α/β receptor signaling or mixed lineage kinase domainlike pseudokinase (MLKL)-dependent necroptotic cell death pathways. Furthermore, radiation chimera experiments reveal how bone marrow from the V154M mutant mice transfer disease to the WT host, whereas the N153S does not, indicating mutation-specific disease outcomes. Moreover, using radiation chimeras we find that T cell lymphopenia depends on T cell-intrinsic expression of the SAVI mutation. Collectively, these mutant mice recapitulate many of the disease features seen in SAVI patients and highlight mutation-specific functions of STING that shed light on the heterogeneity observed in SAVI patients. STING | SAVI | type I interferonopathies | T cells | cell death N ucleic acids are readily detected by nucleic acid sensors that survey cells for signs of infection or tissue damage. Engagement of a diverse collection of RNA and DNA sensors trigger host-defense responses to curb pathogen replication and initiate beneficial repair responses to tissue injury. The DNA sensor cGAS (cGMP-AMP synthase) is a nucleotidyl transferase that detects double-stranded DNA and generates a novel secondmessenger 2′-5′cGMP-AMP (cGAMP). cGAMP binds stimulator of IFN genes (STING), causing its dimerization leading to activation of TBK1/IRF3 and IKK/NF-κB, pathways resulting in the induction of type I IFNs and proinflammatory cytokines, respectively (1). DNases outside cells (DNase I), within the phagolysosomal compartment (DNase II) or cytosol (DNase III/Trex1), ensure that in healthy individuals self nucleic acids do not trigger DNA sensors (2). Inappropriate clearance of DNA and its subsequent detection by DNA sensors underlies the pathogenesis of debilitating human diseases, such as Aicardi-Goutiéres syndrome (3). A subset of Aicardi-Goutiéres syndrome patients have mutations in Trex1, a 3′-5′ exonuclease that degrades DNA, which accumulates from endogenous retroelements (4, 5).Gain-of-function (GOF) mutations in components of RNA and DNA sensing pathways can also result in severe autoinflammatory and autoimmune diseases. Examples include the GOF mutations in DDX58 (RIG-I) and ...

In mice that fail to express the phagolysosomal endonuclease, DNase II, and the type I IFN receptor, excessive accrual of undegraded DNA results in a STING-dependent, TLR-independent inflammatory arthritis. These double knockout (DKO) mice develop additional indications of systemic autoimmunity, including anti-nuclear autoantibodies and splenomegaly, not found in Unc93b1−/− DKO mice and therefore TLR-dependent. The DKO autoantibodies predominantly detect RNA-associated autoantigens, commonly targeted in TLR7-dominated SLE-prone mice. To determine whether an inability of TLR9 to detect endogenous DNA could explain the absence of dsDNA-reactive autoantibodies in DKO mice, we used a novel class of bifunctional autoantibodies, IgM/DNA DVD-Ig™ molecules, to activate B cells through a BCR/TLR9-dependent mechanism. DKO B cells could not respond to the IgM/DNA DVD-Ig™ molecule, despite a normal response to both anti-IgM and CpG ODN 1826. Thus DKO B cells only respond to RNA-associated ligands because DNase II-mediated degradation of self-DNA is required for TLR9 activation.

In recent years, proinflammatory cytokines in the nephritic kidney appear to contribute to the pathogenesis of AGN. The complex inflammatory cytokine network that drives renal pathology is poorly understood. IL-17, the signature cytokine of Th17 cells, which promotes autoimmune pathology in a variety of settings, is beginning to be identified in acute and chronic kidney diseases as well. However, the role of IL-17-mediated renal damage in the nephritic kidney has not been elucidated. Here, with the use of a murine model of experimental AGN, we showed that IL-17RA signaling is critical for the development of renal pathology. Despite normal systemic autoantibody response and glomerular immune-complex deposition, IL-17RA(-/-) mice exhibit a diminished influx of inflammatory cells and kidney-specific expression of IL-17 target genes correlating with disease resistance in AGN. IL-17 enhanced the production of proinflammatory cytokines and chemokines from tECs. Finally, we were able to show that neutralization of IL-17A ameliorated renal pathology in WT mice following AGN. These results clearly demonstrated that IL-17RA signaling significantly contributes to renal tissue injury in experimental AGN and suggest that blocking IL-17RA may be a promising therapeutic strategy for the treatment of proliferative and crescentic glomerulonephritis.

Unraveling of microbial genome data has indicated that two distantly related truncated hemoglobins (trHbs), HbN and HbO, might occur in many species of slow-growing pathogenic mycobacteria. Involvement of HbN in bacterial defense against NO toxicity and nitrosative stress has been proposed. A gene, encoding a putative HbN homolog with conserved features of typical trHbs, has been identified within the genome sequence of fast-growing mycobacterium, Mycobacterium smegmatis. Sequence analysis of M. smegmatis HbN indicated that it is relatively smaller in size and lacks N-terminal pre-A region, carrying 12-residue polar sequence motif that is present in HbN of M. tuberculosis. HbN encoding gene of M. smegmatis was expressed in E. coli as a 12.8 kD homodimeric heme protein that binds oxygen reversibly with high affinity (P 50 $ 0.081 mm Hg) and autooxidizes faster than M. tuberculosis HbN. The circular dichroism spectra indicate that HbN of M. smegmatis and M. tuberculosis are structurally similar. Interestingly, an hmp mutant of E. coli, unable to metabolize nitric oxide, exhibited very low NO uptake activity in the presence of M. smegmatis HbN as compared to HbN of M. tuberculosis. On the basis of cellular heme content, specific nitric oxide dioxygenase (NOD) activity of M. smegmatis HbN was nearly one-third of that from M. tuberculosis. Additionally, the hmp mutant of E. coli, carrying M. smegmatis HbN, exhibited nearly 10-fold lower cell survival under nitrosative stress and nitrite derived reactive nitrogen species as compared to the isogenic strain harboring HbN of M. tuberculosis. Taken together, these results suggest that NO metabolizing activity and protection provided by M. smegmatis HbN against toxicity of NO and reactive nitrogen is significantly lower than HbN of M. tuberculosis. The lower efficiency of M. smegmatis HbN for NO detoxification as compared to M. tuberculosis HbN might be related to different level of NO exposure and nitrosative stress faced by these mycobacteria during their cellular metabolism.

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